Hydantoins that modulate bace-mediated app processing

ABSTRACT

In certain embodiments hydantoin compounds are provided herein that are effective to inhibit BACE activity against APP. Without being bound to a particular theory, it is believed the activity of the hydantoins identified herein appears to be associated with binding to BACE and/or to APP particularly when these moieties form a BACE/APP complex. Accordingly, it is believed the compounds described herein represent a new class of compounds designated herein as APP-Binding-BACE Inhibitors (ABBIs) and provide a new mechanism to modulate APP processing. The hydantoins described herein appear to show improved brain permeability and functional BACE inhibition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 14/928,775, filed onOct. 30, 2015, which is a continuation of U.S. Ser. No. 14/179,310,filed on Feb. 12, 2014, which claims benefit of and priority to U.S.Ser. No. 61/763,830, filed on Feb. 12, 2013, both of which areincorporated herein by reference for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

[Not Applicable]

BACKGROUND

Amyloid beta peptide (Aβ) is a primary component of beta amyloid fibrilsand plaques, which are regarded as having a role in an increasing numberof pathologies. Examples of such pathologies include, but are notlimited to, Alzheimer's disease, Down's syndrome, Parkinson's disease,memory loss (including memory loss associated with Alzheimer's diseaseand Parkinson's disease), attention deficit symptoms (includingattention deficit symptoms associated with Alzheimer's disease,Parkinson's disease, and Down's syndrome), dementia (includingpre-senile dementia, senile dementia, dementia associated withAlzheimer's disease, Parkinson's disease, and Down's syndrome),progressive supranuclear palsy, cortical basal degeneration,neurodegeneration, olfactory impairment (including olfactory impairmentassociated with Alzheimer's disease, Parkinson's disease, and Down'ssyndrome), β-amyloid angiopathy (including cerebral amyloid angiopathy),hereditary cerebral hemorrhage, mild cognitive impairment (“MCI”),glaucoma, amyloidosis, type II diabetes, hemodialysis (β2 microglobulinsand complications arising therefrom), neurodegenerative diseases such asscrapie, bovine spongiform encephalitis, Creutzfeld Jakob disease,traumatic brain injury and the like.

Aβ peptides are short peptides that are produced by proteolysis of thetransmembrane protein called amyloid precursor protein (“APP”). Aβpeptides are made from the cleavage of APP by β-secretase activity at aposition near the N-terminus of Aβ, and by gamma secretase activity at aposition near the C-terminus of Aβ. (APP is also cleaved by α-secretaseactivity, resulting in the secreted, non-amyloidogenic fragment known assoluble APPα). Beta site APP Cleaving Enzyme (“BACE-1”) is regarded asthe primary aspartyl protease responsible for the production of Aβ byβ-secretase activity. The inhibition of BACE-1 has been shown to inhibitthe production of Aβ.

Alzheimer's disease (AD) is estimated to afflict more than 20 millionpeople worldwide and is believed to be the most common cause ofdementia. As the World population ages, the number of people withAlzheimer's disease (AD, currently approximately 5.4 million in theUnited States, will continue to rise. Alzheimer's is a neurodegenerativedisease associated with progressive dementia and memory loss. Two keycharacteristics of AD are the accumulation of extracellular depositscontaining aggregated Aβ peptide and neuronal synaptic loss in the AD inspecific brain regions. Although AD pathogenesis is complex, compellinggenetic and biochemical evidence suggest that overproduction of Aβ, orfailure to clear this peptide is the earliest event in the amyloidcascade that lead to AD primarily through amyloid deposition, which ispresumed to be involved in neurofibrillary tangle formation, neuronaldysfunction and microglia activation, that characterize AD-affectedbrain tissues.

The accumulation of Aβ is considered to be the earliest event in acomplex cascade that leads to neurodegeneration, as discerned fromcompelling genetic and biochemical evidence. The amyloid cascadehypothesis (Hardy and Allsop (1991) Trends Pharmacol. Sci., 12: 383-388;Selkoe (1996)J. Biol. Chem., 271: 18295-18298; Hardy (1997) TrendsNeurosci., 20: 154-159; Hardy and Selkoe (2002) Science, 297: 353-356)states that overproduction of Aβ, or failure to clear this peptide,leads to AD, primarily through amyloid deposition, which is presumed tobe involved in neurofibrillary tangle formation, neuronal dysfunction,and microglia activation, that are hallmarks of AD-affected braintissues (Busciglio et al. (1995) Neuron, 14: 879-888; Gotz et al. (1995)EMBO J., 14: 1304-1313; Lewis et al. (2001) Science, 293: 1487-1491;Hardy et al. (1985) Nat Neurosci., 1: 355-358).

Considering the causative role of Aβ in AD etiology, novel therapeuticstrategies that lower Aβ levels or prevent the formation of theneurotoxic Aβ species have been suggested as a method to prevent or slowthe progression of the disease. Indeed, the major focus over the lastdecade has been to inhibit brain Aβ production and aggregation, toincrease parenchymal Aβ clearance, and to interfere with Aβ-induced celldeath.

The sequential cleavage of APP by membrane-bound proteases β-secretaseand γ-secretase results in the formation of Aβ. A competing proteolyticpathway to the β-secretase pathway, the α-secretase pathway, results incleavage of APP within the Aβ domain, thereby precluding the generationof Aβ (Selkoe (2001) Physiol. Rev., 81: 741-766; Hussain et al. (1999)Mol. Cell. Neurosci., 14: 419-427; Sinha et al. (1999) Nature, 402:537-540; Vassar et al. (1999) Science, 286: 735-741). The β-Site APPcleavage enzyme-1 (BACE1) was identified as the major β-secretaseactivity that mediates the first cleavage of APP in the β-amyloidogenicpathway (Id.).

BACE1 is a 501 amino acid protein that bears homology to eukaryoticaspartic proteases, especially from the pepsin family (Yan et al. (1999)Nature, 402: 533-537). Similar to other aspartic proteases, BACE1 issynthesized as a zymogen with a pro-domain that is cleaved by furin torelease the mature protein. BACE1 is a type-I transmembrane protein witha luminal active site that cleaves APP to release an ectodomain (sAPPβ)into the extracellular space. The remaining C-terminal fragment (CTF)undergoes further cleavage by γ-secretase, leading to the release of Aβand the APP intracellular C-terminal domain (AICD).

The presenilins have been proposed to be the major enzymatic componentof γ-secretase, whose imprecise cleavage of APP produces a spectrum ofAβ peptides varying in length by a few amino acids at the C-terminus.The majority of Aβ normally ends at amino acid 40 (Aβ40), but the42-amino acid variant (Aβ42) has been shown to be more susceptible toaggregation, and has been hypothesized to nucleate senile plaqueformation. The modulation of the γ-secretase can also lead to increasein the 38-amino acid variant (Aβ38). The competing α-secretase pathwayis the result of sequential cleavages by α- and γ-secretase. Threemetalloproteases of the disintegrin and metalloprotease family (ADAM 9,10, and 17) have been proposed as candidates for the α-secretaseactivity, which cleaves APP at position 16 within the Aβ sequence. Usingoverexpression experiments, ADAM-10 has been shown to be the likelyα-secretase for cleavage of APP (Vassar (2002) Adv. Drug Deliv. Rev.,54: 1589-1602; Buxbaum et al. (1998) J. Biol. Chem., 273: 27765-27767;Koike et al. (1999) Biochem. J., 343(Pt 2): 371-375). This cleavage alsoreleases an ectodomain (sAPPα), which displays neuroprotective functions(Lammich et al. (1999) Proc. Natl. Acad. Sci. USA, 96: 3922-3927).Subsequent cleavage of the 83-amino acid CTF (C83) releases p3, which isnon-amyloidogenic, and AICD (Furukawa et al. (1996) J. Neurochem., 67:1882-1896). The functions of these fragments are not fully elucidated,although AICD is hypothesized to mediate intracellular signaling.

Research clarifying the metabolic pathways that regulate the productionof Aβ from the Amyloid Precursor Protein (APP) indicates that thesecretases that produce Aβ are good therapeutic targets, sinceinhibition of either β- or γ-secretase limits Aβ production. The factthat β-secretase initiates APP processing, and thus serves as the ratelimiting step in production of Aβ, its inhibition has attracted effortsby many research groups. Examples from the patent literature are growingand include, for example, WO2006009653, WO2007005404, WO2007005366,WO2007038271, WO2007016012, US2005/0282826, US2007072925, WO2007149033,WO2007145568, WO2007145569, WO2007145570, WO2007145571, WO2007114771,US20070299087, WO2005/016876, WO2005/014540, WO2005/058311,WO2006/065277, WO2006/014762, WO2006/014944, WO2006/138195,WO2006/138264, WO2006/138192, WO2006/138217, WO2007/050721,WO2007/053506, WO2007/146225, WO2006/138230, WO2006/138265,WO2006/138266, WO2007/053506, WO2007/146225, WO2008/073365,WO2008/073370, WO2008/103351, US2009/041201, US2009/041202, andWO2010/047372.

One limitation of protease inhibitory strategies is the inhibition ofcleavage of all substrates of a given targeted protease, such as BACE orthe γ-secretase complex. In the case of γ-secretase, substrates otherthan APP, such as Notch, raise concerns for potential side effects ofγ-secretase inhibition, and the recent failure of the γ-secretaseinhibitor. Problems associated with the use of semagacestat, serve toreinforce such concerns.

BACE is a key enzyme involved in processing of APP leading to theproduction of Aβ42 and the Alzheimer's disease (AD) pathology. BACE-1(also called BACE) has become a popular research area since itsdiscovery, and has perhaps surpassed γ-secretase as the most promisingtarget for pharmaceutical research. A problem with γ-secretase as atarget is its known cleavage of Notch, which serves important functionsin neuronal development. Presenilin knockout mice demonstrated abnormalsomitogenesis and axial skeletal development with shortened body length,as well as cerebral hemorrhages (Shen et al. (1997) Cell, 89: 629-639;Wong et al. (1997) Nature, 387: 288-292). In contrast, several groupsreported that BACE1 knockout mice are healthy and show no signs ofadverse effect (Luo et al. (2001) Nat. Neurosci., 4: 231-232; Roberds etal. (2001) Hum. Mol. Genet., 10: 1317-1324), while one group noticedsubtle neurochemical deficits and behavioral changes in otherwise viableand fertile mice (Harrison et al. (2003) Mol. Cell Neurosci., 24:646-655). Although recent studies have shown that BACE1 knockout miceexhibit hypomyelination of peripheral nerves (Willem et al. (2006)Science, 314: 664-666), the consequences of BACE1 inhibition in adultanimals, where myelination has already taken place, are unclear.Recently BACE1 has been reported to cleave multiple substrates,including ST6Gal I, PSGL-1, subunits of voltage-gated sodium channels,APP-like proteins (APLPs), LDL receptor related protein (LRP) and, mostrecently, type III neuregulin 1 (NRG1) (Willem et al. (2006) Science,314: 664-666; Hu et al. (2006) Nat. Neurosci., 9: 1520-1525). Theconsequences of inhibiting BACE1 directly are therefore not yet fullyunderstood.

Molecular modeling (Sauder et al. (2000) J. Mol. Biol., 300: 241-248)and subsequent X-ray crystallography (Hong et al. (2000) Science, 290:150-153; Maillard et al. (2007) J. Med. Chem., 50: 776-781) of theBACE-1 active site complexed with a transition-state inhibitor providedcrucial information about BACE-1-substrate interactions. Structurally,the BACE-1 active site is more open and less hydrophobic than otheraspartyl proteases, making development of effective in vivo BACEinhibitor candidates difficult. While a there is a large drug discoveryeffort focused on development of direct BACE inhibitors, none so farhave advanced significantly in clinical testing.

A few BACE inhibitors such as LY2811376 and CTS21166 entered clinicaltesting, but did not go forward beyond Phase-1 due to safety reasons.The discovery of other physiological substrates of BACE raises a majorconcern in the clinical development of BACE inhibitors or BACEmodulators and could be a significant roadblock in advancement of theseinhibitors as a therapy for the disease.

SUMMARY

In certain embodiments hydantoin compounds are provided herein that areeffective to inhibit BACE activity against APP. Without being bound to aparticular theory, it is believed the activity of the hydantoinsidentified herein appears to be associated with binding to BACE and/orto APP particularly when these moieties form a BACE/APP complex.Accordingly, it is believed the compounds described herein represent anew class of compounds designated herein as APP-Binding-BACE Inhibitors(ABBIs) and provide a new mechanism to modulate APP processing. Thehydantoins described herein appear to show improved brain permeabilityand functional BACE inhibition.

In various aspects, the invention(s) contemplated herein may include,but need not be limited to, any one or more of the followingembodiments:

Embodiment 1

A compound according to the formula:

where M is

R⁷ is selected from the group consisting of C═O, C═S, C—NH₂, and C═NH,and the bond represented by the wavy line is a single bond when R⁷ isC═O, C═S, or C═NH, and a double bond when R⁷ is C—NH₂; R⁸ and R⁹ areindependently selected from the group consisting of H, alkyl,cycloalkyl, and aryl, provided that when the bond represented by thewavy line is a double bond, then R⁹ is absent; R⁰ is selected from thegroup consisting of aryl, substituted aryl, disubstituted aryl,heteroaryl, substituted heteroaryl, disubstituted heteroaryl, alkyl,haloalkyl, cycloalkyl, alkenyl, and alkynyl; X¹ is selected from thegroup consisting of C-halogen (e.g., Cl or F), CH, and N; A is methyl orH; R⁵ and R⁶ are independently selected from halogen, H, alkyl, aryl,trichloromethyl, and trifluoromethyl; R³ and R⁴ are independently absentor selected from the group consisting of alkyl, cycloalkyl, alkoxy,thioalky; and when X¹ is C, then R⁰ is not phenyl monosubstituted at thepara position with —OCHF₂, or a pharmaceutically acceptable saltthereof, a tautomer thereof, a pharmaceutically acceptable salt of atautomer thereof, an enantiomer thereof, or a pharmaceuticallyacceptable salt of an enantiomer thereof.

Embodiment 2

The compound of embodiment 1, wherein said compound is a compoundaccording to the formula:

Embodiment 3

The compound of embodiment 1, wherein said compound is a compoundaccording to the Formula:

Embodiment 4

The compound according to any one of embodiments 1-3, wherein R⁵ and R⁶are independently selected from halogen, H, alkyl, trichloromethyl, andtrifluoromethyl.

Embodiment 5

The compound according to any one of embodiments 1-4, wherein: X¹ isselected from the group consisting of CH, and N; and R⁵ and R⁶ areindependently selected halogen.

Embodiment 6

The compound according to any one of embodiments 1-5, wherein R⁷ isC═NH.

Embodiment 7

The compound according to any one of embodiments 1-5, wherein R⁷ is C═O.

Embodiment 8

The compound of embodiment 6, wherein said compound is a compound havingthe formula:

Embodiment 9

The compound of embodiment 8, wherein R⁵ and R⁶ are independentlyselected halogens.

Embodiment 10

The compound of embodiment 9, wherein R⁵ and R⁶ are the same halogen.

Embodiment 11

The compound of embodiment 9, wherein R⁵ and R⁶ are both F.

Embodiment 12

The compound of embodiment 7, wherein said compound is a compound ofhaving the formula:

Embodiment 13

The compound of embodiment 7, wherein said compound is a compound of theformula:

Embodiment 14

The compound of embodiment 7, wherein said compound is a compound of theformula:

Embodiment 15

The compound of embodiment 3, wherein R⁷ is C═S.

Embodiment 16

The compound according to any one of embodiments 1-5, wherein R⁷ isC—NH₂ and said compound is a compound having the formula:

Embodiment 17

The compound of embodiment 16, wherein said compound is a compound ofFormula:

where R¹ and R² are independently absent or selected from the groupconsisting of alkyl, haloalkyl, cycloalkyl, alkenyl, alkynyl, alkoxy,thioalkyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; and X², Y, and Z are independently CH or N.

Embodiment 18

The compound according to any one of embodiments 1-17, wherein R⁵ and R⁶are different halogens.

Embodiment 19

The compound according to any one of embodiments 1-17, wherein R⁵ and R⁶are the same halogen.

Embodiment 20

The compound according to any one of embodiments 1-19, wherein R⁵ and R⁶are independently Cl or F.

Embodiment 21

The compound of embodiment 17, wherein said compound is a compoundhaving the formula:

Embodiment 22

The compound according to any one of embodiments 1-7 and 15-21, whereinX¹ is CH.

Embodiment 23

The compound according to any one of embodiments 1-7 and 15-22, whereinR⁸ is H or CH₃.

Embodiment 24

The compound of embodiment 3, wherein said compound is a compound havingthe Formula:

Embodiment 25

The compound of embodiment 3, wherein said compound is a compound havingthe Formula:

Embodiment 26

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 27

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 28

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 29

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 30

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 31

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 32

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 33

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 34

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 35

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 36

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 37

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 38

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 39

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 40

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 41

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 42

The compound of embodiment 3, wherein said compound is a compound havingthe formula:

Embodiment 43

A compound of Formula:

or a pharmaceutically acceptable salt thereof, a tautomer thereof, apharmaceutically acceptable salt of a tautomer thereof, an enantiomerthereof, or a pharmaceutically acceptable salt of an enantiomer thereof.

Embodiment 44

The compound according to any one of embodiments 1-43, wherein saidcompound is a substantially pure S enantiomer.

Embodiment 45

The compound according to any one of embodiments 1-43, wherein saidcompound is a substantially pure R enantiomer.

Embodiment 46

The compound according to any one of embodiments 1-45, wherein saidcompound binds to APP and/or to the enzyme BACE and/or to an APP/BACEcomplex.

Embodiment 47

The compound according to any one of embodiments 1-45, wherein saidcompound binds to APP and inhibits the enzyme BACE.

Embodiment 48

A pharmaceutical formulation including a pharmaceutically acceptablecarrier and a compound according to any one of embodiments 1-47.

Embodiment 49

The formulation of embodiment 48, wherein said formulation is compoundedfor administration via a route selected from the group consisting oforal delivery, isophoretic delivery, transdermal delivery, parenteraldelivery, aerosol administration, administration via inhalation,intravenous administration, and rectal administration.

Embodiment 50

The formulation of embodiment 48, wherein said formulation is compoundedfor oral administration.

Embodiment 51

The formulation of embodiment 48, wherein said formulation is sterile.

Embodiment 52

The formulation according to any one of embodiments 48-51, wherein saidformulation is a unit dosage formulation.

Embodiment 53

A method of preventing or delaying the onset of a pre-Alzheimer'scondition and/or cognitive dysfunction, and/or ameliorating one or moresymptoms of a pre-Alzheimer's condition and/or cognitive dysfunction, orpreventing or delaying the progression of a pre-Alzheimer's condition orcognitive dysfunction to Alzheimer's disease, said method including:administering to a subject in need thereof a compound according to anyone of embodiments 1-47, or formulation according to any one ofembodiments 48-52 in an amount sufficient to prevent or delay the onsetof a pre-Alzheimer's cognitive dysfunction, and/or to ameliorate one ormore symptoms of a pre-Alzheimer's cognitive dysfunction, and/or toprevent or delay the progression of a pre-Alzheimer's cognitivedysfunction to Alzheimer's disease.

Embodiment 54

The method of embodiment 53, wherein said method is a method ofpreventing or delaying the transition from a cognitively asymptomaticpre-Alzheimer's condition to a pre-Alzheimer's cognitive dysfunction.

Embodiment 55

The method of embodiment 53, wherein said method is a method ofpreventing or delaying the onset of a pre-Alzheimer's cognitivedysfunction.

Embodiment 56

The method of embodiment 53, wherein said method includes amelioratingone or more symptoms of a pre-Alzheimer's cognitive dysfunction.

Embodiment 57

The method of embodiment 53, wherein said method includes preventing ordelaying the progression of a pre-Alzheimer's cognitive dysfunction toAlzheimer's disease.

Embodiment 58

The method according to any one of embodiments 53-57, wherein saidsubject is a human.

Embodiment 59

The method according to any one of embodiments 53-58, wherein saidsubject exhibits biomarker positivity of Aβ in a clinically normal humansubject age 50 or older.

Embodiment 60

The method according to any one of embodiments 53-58, wherein saidsubject exhibits asymptomatic cerebral amyloidosis.

Embodiment 61

The method according to any one of embodiments 53-58, wherein saidsubject exhibits cerebral amyloidosis in combination with downstreamneurodegeneration.

Embodiment 62

The method according to any one of embodiments 53-58, wherein saidsubject exhibits cerebral amyloidosis in combination with downstreamneurodegeneration and subtle cognitive/behavioral decline.

Embodiment 63

The method according to any one of embodiments 61-62, wherein saiddownstream neurodegeneration is determined by one or more elevatedmarkers of neuronal injury selected from the group consisting of tau,and FDG uptake.

Embodiment 64

The method according to any one of embodiments 60-63, wherein saidcerebral amyloidosis is determined by PET, or CSF analysis, andstructural MRI (sMRI).

Embodiment 65

The method according to any one of embodiments 53-64, wherein saidsubject is a subject diagnosed with mild cognitive impairment.

Embodiment 66

The method according to any one of embodiments 53-65, wherein saidsubject shows a clinical dementia rating above zero and below about 1.5.

Embodiment 67

The method according to any one of embodiments 53-66, wherein thesubject is human.

Embodiment 68

The method according to any one of embodiments 53-67, wherein thesubject is at risk of developing Alzheimer's disease.

Embodiment 69

The method according to any one of embodiments 53-68, wherein thesubject has a familial risk for having Alzheimer's disease.

Embodiment 70

The method according to any one of embodiments 53-68, wherein thesubject has a familial Alzheimer's disease (FAD) mutation.

Embodiment 71

The method according to any one of embodiments 53-68, wherein thesubject has the APOE E4 allele.

Embodiment 72

The method according to any one of embodiments 53-71, whereinadministration of said compound delays or prevents the progression ofMCI to Alzheimer's disease.

Embodiment 73

The method according to any one of embodiments 53-72, wherein thesubject is free of and does not have genetic risk factors of Parkinson'sdisease or schizophrenia.

Embodiment 74

The method according to any one of embodiments 53-72, wherein thesubject is not diagnosed as having or at risk for Parkinson's disease orschizophrenia.

Embodiment 75

The method according to any one of embodiments 53-72, wherein thesubject is not diagnosed as at risk for a neurological disease ordisorder other than Alzheimer's disease.

Embodiment 76

The method according to any one of embodiments 53-75, wherein saidadministration produces a reduction in the CSF of levels of one or morecomponents selected from the group consisting of Aβ42, sAPPβ, total-Tau(tTau), phospho-Tau (pTau), APPneo, soluble Aβ40, pTau/Aβ42 ratio andtTau/Aβ42 ratio, and/or an increase in the CSF of levels of one or morecomponents selected from the group consisting of Aβ42/Aβ40 ratio,Aβ42/Aβ38 ratio, sAPPα, sAPPα/sAPPβ ratio, sAPPα/Aβ40 ratio, andsAPPα/Aβ42 ratio.

Embodiment 77

The method according to any one of embodiments 53-76, wherein saidadministration produces a reduction of the plaque load in the brain ofthe subject.

Embodiment 78

The method according to any one of embodiments 53-76, wherein saidadministration produces a reduction in the rate of plaque formation inthe brain of the subject.

Embodiment 79

The method according to any one of embodiments 53-76, wherein saidadministration produces an improvement in the cognitive abilities of thesubject.

Embodiment 80

The method according to any one of embodiments 53-76, wherein saidadministration produces an improvement in, a stabilization of, or areduction in the rate of decline of the clinical dementia rating (CDR)of the subject.

Embodiment 81

The method according to any one of embodiments 53-76, wherein thesubject is a human and said administration produces a perceivedimprovement in quality of life by the human.

Embodiment 82

The method according to any one of embodiments 53-81, wherein thecompound or formulation is administered via a route selected from thegroup consisting of oral delivery, isophoretic delivery, transdermaldelivery, parenteral delivery, aerosol administration, administrationvia inhalation, intravenous administration, and rectal administration.

Embodiment 83

The method according to any one of embodiments 53-81, wherein thecompound or formulation is administered orally.

Embodiment 84

The method according to any one of embodiments 53-83, wherein theadministering is over a period of at least three weeks.

Embodiment 85

The method according to any one of embodiments 53-83, wherein theadministering is over a period of at least 6 months.

Embodiment 86

A method of ameliorating one or more symptoms of Alzheimer's disease,and/or reversing Alzheimer's disease, and/or reducing the rate ofprogression of Alzheimer's disease, said method including: administeringto a subject in need thereof a compound according to any one ofembodiments 1-47, or formulation according to any one of embodiments48-52 in an amount sufficient to ameliorate one or more symptoms ofAlzheimer's disease, and/or to reverse Alzheimer's disease, and/or toreduce the rate of progression of Alzheimer's disease.

Embodiment 87

The method of embodiment 86, wherein said subject is a human.

Embodiment 88

The method of embodiment 87, wherein said subject is a human at least 50years old.

Embodiment 89

The method according to any one of embodiments 86-88, wherein saidsubject is diagnosed with early stage Alzheimer's disease.

Embodiment 90

The method according to any one of embodiments 86-88, wherein saidsubject is diagnosed with mid-stage Alzheimer's disease.

Embodiment 91

The method according to any one of embodiments 86-88, wherein saidsubject is diagnosed with late-stage Alzheimer's disease.

Embodiment 92

The method according to any one of embodiments 86-91, wherein saidadministering reduces the severity of Alzheimer's disease.

Embodiment 93

The method according to any one of embodiments 86-91, wherein saidadministering ameliorates one or more symptoms of Alzheimer's disease.

Embodiment 94

The method according to any one of embodiments 86-91, wherein saidadministering reduces the rate of progression of Alzheimer's disease.

Embodiment 95

The method according to any one of embodiments 86-94, wherein saidadministering results in a reduction in the CSF of levels of one or morecomponents selected from the group consisting of Aβ42, sAPPβ, total-Tau(tTau), phospho-Tau (pTau), APPneo, soluble Aβ40, pTau/Aβ42 ratio andtTau/Aβ42 ratio, and/or an increase in the CSF of levels of one or morecomponents selected from the group consisting of Aβ42/Aβ40 ratio,Aβ42/Aβ38 ratio, sAPPα, sAPPα/sAPPβ ratio, sAPPα/Aβ40 ratio, andsAPPα/Aβ42 ratio. is a method of preventing or delaying the transitionfrom a cognitively asymptomatic pre-Alzheimer's condition to apre-Alzheimer's cognitive dysfunction.

Embodiment 96

The method according to any one of embodiments 86-95, wherein saidadministration produces a reduction of the plaque load in the brain ofthe subject.

Embodiment 97

The method according to any one of embodiments 86-95, wherein saidadministration produces a reduction in the rate of plaque formation inthe brain of the subject.

Embodiment 98

The method according to any one of embodiments 86-95, wherein saidadministration produces an improvement in the cognitive abilities of thesubject.

Embodiment 99

The method according to any one of embodiments 86-95, wherein saidadministration produces an improvement in, a stabilization of, or areduction in the rate of decline of the clinical dementia rating (CDR)of the subject.

Embodiment 100

The method according to any one of embodiments 86-95, wherein thesubject is a human and said administration produces a perceivedimprovement in quality of life by the human.

Embodiment 101

The method according to any one of embodiments 86-95, wherein saidadministering results in reduced cerebral amyloidosis and/or downstreamneurodegeneration.

Embodiment 102

The method of embodiment 101, wherein said downstream neurodegenerationis determined by one or more markers of neuronal injury selected fromthe group consisting of tau, FDG uptake, decrease in sAPPalpha, increasein sAPPbeta, and Abeta.

Embodiment 103

The method according to any one of embodiments 101-102, wherein saidcerebral amyloidosis is determined by PET using amyloid/tau bindingagents, CSF analysis. and structural MRI (sMRI).

Embodiment 104

The method according to any one of embodiments 86-103, wherein saidsubject shows a clinical dementia rating indicative of Alzheimer'sdisease.

Embodiment 105

The method according to any one of embodiments 86-104, wherein thesubject has a familial risk for having Alzheimer's disease.

Embodiment 106

The method according to any one of embodiments 86-105, wherein thesubject has a familial Alzheimer's disease (FAD) mutation.

Embodiment 107

The method according to any one of embodiments 86-105, wherein thesubject has the APOE ε4 allele.

Embodiment 108

The method according to any one of embodiments 86-107, wherein thesubject is free of and does not have genetic risk factors of Parkinson'sdisease or schizophrenia.

Embodiment 109

The method according to any one of embodiments 86-107, wherein thesubject is not diagnosed as having or at risk for Parkinson's disease orschizophrenia.

Embodiment 110

The method according to any one of embodiments 86-109, wherein thesubject does not have a neurological disease or disorder other thanAlzheimer's disease.

Embodiment 111

The method according to any one of embodiments 86-110, wherein thesubject is not diagnosed as having or at risk for a neurological diseaseor disorder other than Alzheimer's disease.

Embodiment 112

The method according to any one of embodiments 86-111, wherein thecompound is administered via a route selected from the group consistingof oral delivery, isophoretic delivery, transdermal delivery, parenteraldelivery, aerosol administration, administration via inhalation,intravenous administration, and rectal administration.

Embodiment 113

The method according to any one of embodiments 86-112, wherein thecompound is formulated for administration via a route selected from thegroup consisting of oral delivery, isophoretic delivery, transdermaldelivery, parenteral delivery, aerosol administration, administrationvia inhalation, intravenous administration, and rectal administration.

Embodiment 114

The method according to any one of embodiments 86-113, wherein thecompound is administered orally.

Embodiment 115

The method according to any one of embodiments 86-114, wherein theadministering is over a period of at least three weeks.

Embodiment 116

The method according to any one of embodiments 86-114, wherein theadministering is over a period of at least 6 months.

Embodiment 117

The method according to any one of embodiments 53-116, wherein saidcompound is administered in combination with one or more agents selectedfrom the group consisting of disulfiram and/or analogues thereof,honokiol and/or analogues thereof, tropisetron and/or analogues thereof,nimetazepam and/or analogues thereof, tropinol-esters and/or relatedesters and/or analogues thereof, TrkA kinase inhibitors (e.g.,ADDN-1351) and/or analogues thereof, D2 receptor agonists,alpha1-adrenergic receptor antagonists, and APP-specific BACE Inhibitorsincluding, but not limited to galangin, a galangin prodrug, rutin, arutin prodrug, and other flavonoids and flavonoid prodrugs.

Embodiment 118

The method of embodiment 117, wherein said compound is administered incombination with tropisetron.

Embodiment 119

A method of slowing the progression, stopping, or reversing age-relatedmacular degeneration (AMD) in a mammal, said method includingadministering to said mammal a compound according to any one ofembodiments 1-47, or formulation according to any one of embodiments48-52 in an amount sufficient to slow the progression, stop, or reverseage-related macular degeneration in said mammal.

Embodiment 120

A method for the treatment of a disease or disorder associated with BACEactivity in a subject in need thereof, wherein said method includesproviding to said subject a therapeutically effective amount of acompound according to any one of embodiments 1-47, or formulationaccording to any one of embodiments 48-52.

Embodiment 121

The method of embodiment 120, wherein said disease or disorder isselected from the group consisting of Alzheimer's disease; cognitiveimpairment, Down's Syndrome, HCHWA-D, cognitive decline, seniledementia, cerebral amyloid angiopathy, and a neurodegenerative disorder.

Embodiment 122

The method of embodiment 121, wherein said disease or disorder ischaracterized by the production of amyloid deposits and/orneurofibrillary tangles.

Embodiment 123

A kit including one or more containers containing a compound accordingto any one of embodiments 1-47, or formulation according to any one ofembodiments 48-52.

Embodiment 124

The kit of embodiment 123, wherein said kit further includes a secondagent selected from the group consisting of disulfiram and/or analoguesthereof, honokiol and/or analogues thereof, tropisetron and/or analoguesthereof, nimetazepam and/or analogues thereof, tropinol-esters and/orrelated esters and/or analogues thereof, TrkA kinase inhibitors (e.g.,ADDN-1351) and/or analogues thereof, D2 receptor agonists,alpha1-adrenergic receptor antagonists, and APP-specific BACE Inhibitorsincluding, but not limited to galangin, a galangin prodrug, rutin, arutin prodrug, and other flavonoids and flavonoid prodrugs.

Embodiment 125

The kit of embodiment 124, wherein said second agent is tropisetron.

Embodiment 126

The kit according to any one of embodiments 123-125, further includinginstructional materials teaching dosages and treatment regimen for theactive agents contained in the kit.

Embodiment 127

The compounds, methods, or kits according to any one of embodiments1-126, wherein said embodiments expressly exclude FAH-2.

Embodiment 128

The compounds, methods, or kits according to any one of embodiments1-127, wherein said embodiments expressly exclude FAH-3.

Embodiment 129

The compounds, methods, or kits according to any one of embodiments1-128, wherein said embodiments expressly exclude FAH-1.

Embodiment 130

The compounds, methods, or kits according to any one of embodiments1-129, wherein said embodiments expressly exclude FAH-4.

Embodiment 131

The compounds, methods, or kits according to any one of embodiments1-130, wherein said embodiments expressly exclude FAH-5.

Embodiment 132

The compounds, methods, or kits according to any one of embodiments1-131, wherein said embodiments expressly exclude FAH-6.

Embodiment 133

The compounds, methods, or kits according to any one of embodiments1-132, wherein said embodiments expressly exclude FAH-7.

Embodiment 134

The compounds, methods, or kits according to any one of embodiments1-133, wherein said embodiments expressly exclude FAH-8.

Embodiment 135

The compounds, methods, or kits according to any one of embodiments1-134, wherein said embodiments expressly exclude FAH-10

Embodiment 136

The compounds, methods, or kits according to any one of embodiments1-135, wherein said embodiments expressly exclude FAH-11.

Embodiment 137

The compounds, methods, or kits according to any one of embodiments1-136, wherein said embodiments expressly exclude FAH-12.

Embodiment 138

The compounds, methods, or kits according to any one of embodiments1-137, wherein said embodiments expressly exclude FAH-13.

Embodiment 139

The compounds, methods, or kits according to any one of embodiments1-138, wherein said embodiments expressly exclude FAH-14.

Embodiment 140

The compounds, methods, or kits according to any one of embodiments1-139, wherein said embodiments expressly exclude FAH-15.

Embodiment 141

The compounds, methods, or kits according to any one of embodiments1-140, wherein said embodiments expressly exclude FAH-17.

Embodiment 142

The compounds, methods, or kits according to any one of embodiments1-141, wherein said embodiments expressly exclude FAH-19.

Embodiment 143

The compounds, methods, or kits according to any one of embodiments1-142, wherein said embodiments expressly exclude FAH-22.

Embodiment 144

The compounds, methods, or kits according to any one of embodiments1-143, wherein said embodiments expressly exclude FAH-23.

Embodiment 145

The compounds, methods, or kits according to any one of embodiments1-144, wherein said embodiments expressly exclude FAH-25.

Embodiment 146

The compounds, methods, or kits according to any one of embodiments1-145, wherein said embodiments expressly exclude FAH-27.

Embodiment 147

The compounds, methods, or kits according to any one of embodiments1-146, wherein said embodiments expressly exclude FAH-28.

Embodiment 148

The compounds, methods, or kits according to any one of embodiments1-147, wherein the compound(s) described herein (or a tautomer orstereoisomer thereof; or pharmaceutically acceptable salt or solvate ofsaid compound, said stereoisomer, or said tautomer) are administered toa subject not diagnosed with or under treatment for convulsions and/orepilepsy.

Embodiment 149

The compounds, methods, or kits according to any one of embodiments1-148 where the compound(s) described herein (or a tautomer orstereoisomer thereof; or pharmaceutically acceptable salt or solvate ofsaid compound, said stereoisomer, or said tautomer) are administered toa subject not subject to, and/or diagnosed with, and/or under treatmentfor one or more of the following: arrhythmia, epilepsy, neurosurgery,peripheral neuropathy, rheumatoid arthritis, seizure prevention,seizures, status epilepticus, and/or trigeminal neuralgia.

Definitions

Unless otherwise indicated, reference to a compound (e.g., to ahydantoins as described herein) should be construed broadly to includepharmaceutically acceptable salts, prodrugs, tautomers, alternate solidforms, non-covalent complexes, and combinations thereof, of a chemicalentity of the depicted structure or chemical name.

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium. Accordingly, isotopically labeled compounds are within thescope of this invention.

A pharmaceutically acceptable salt is any salt of the parent compoundthat is suitable for administration to an animal or human. Apharmaceutically acceptable salt also refers to any salt which may formin vivo as a result of administration of an acid, another salt, or aprodrug which is converted into an acid or salt. A salt comprises one ormore ionic forms of the compound, such as a conjugate acid or base,associated with one or more corresponding counterions. Salts can formfrom or incorporate one or more deprotonated acidic groups (e.g.carboxylic acids), one or more protonated basic groups (e.g. amines), orboth (e.g. zwitterions).

A prodrug is a compound that is converted to a therapeutically activecompound after administration. For example, conversion may occur byhydrolysis of an ester group, such as a C₁-C₆ alkyl ester of thecarboxylic acid group of the present compounds, or some otherbiologically labile group. Prodrug preparation is well known in the art.For example, “Prodrugs and Drug Delivery Systems,” which is a chapter inRichard B. Silverman, Organic Chemistry of Drug Design and Drug Action,2d Ed., Elsevier Academic Press: Amsterdam, 2004, pp. 496-557, providesfurther detail on the subject.

Tautomers are isomers that are in equilibrium with one another. Forexample, tautomers may be related by transfer of a proton, hydrogenatom, or hydride ion.

Unless stereochemistry is explicitly depicted, a structure is intendedto include every possible stereoisomer, both pure or in any possiblemixture.

Alternate solid forms are different solid forms than those that mayresult from practicing the procedures described herein. For example,alternate solid forms may be polymorphs, different kinds of amorphoussolid forms, glasses, and the like. In various embodiments alternatesolid forms of any of the compounds described herein are contemplated.

In general, “substituted” refers to an organic group as defined below(e.g., an alkyl group) in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms. Substituted groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Thus, a substitutedgroup will be substituted with one or more substituents, unlessotherwise specified. In some embodiments, a substituted group issubstituted with 1, 2, 3, 4, 5, or 6 substituents. Examples ofsubstituent groups include: halogens (i.e., F, Cl, Br, and I);hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy,heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo);carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines;aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls;sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones;azides; amides; ureas; amidines; guanidines; enamines; imides;isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitrogroups; nitriles (i.e., CN), and the like.

The term “alkyl” refers to and covers any and all groups that are knownas normal alkyl, branched-chain alkyl, cycloalkyl and alsocycloalkyl-alkyl. Illustrative alkyl groups include, but are not limitedto methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,t-butyl, octyl, and decyl. The term “cycloalkyl” refers to cyclic,including polycyclic, saturated hydrocarbyl groups. Examples include,but are not limited to cyclopentyl, cyclohexyl, dicyclopentyl, norbomyl,octahydronapthyl, and spiro[3.4]octyl. In certain embodiments, alkylgroups contain 1-12 carbon atoms (C1-12 alkyl), or 1-9 carbon atoms(C₁₋₉ alkyl), or 1-6 carbon atoms (C₁₋₆ alkyl), or 1-5 carbon atoms(C₁₋₅ alkyl), or carbon atoms (C₁₋₄ alkyl), or 1-3 carbon atoms (C₁₋₃alkyl), or 1-2 carbon atoms (C₁₋₂ alkyl).

By way of example, the term “C₁₋₆ alkyl group” refers to a straightchain or branched chain alkyl group having 1 to 6 carbon atoms, and maybe exemplified by a methyl group, an ethyl group, an n-propyl group, anisopropyl group, an n-butyl group, an isobutyl group, a tert-butylgroup, a sec-butyl group, an n-pentyl group, a tert-amyl group, a3-methylbutyl group, a neopentyl group, and an n-hexyl group.

The term “alkoxy” as used herein means an alkyl group bound through asingle, terminal oxygen atom. An “alkoxy” group may be represented as—O-alkyl where alkyl is as defined above. The term “aryloxy” is used ina similar fashion, and may be represented as —O-aryl, with aryl asdefined below. The term “hydroxy” refers to —OH.

Similarly, the term “alkylthio” as used herein means an alkyl groupbound through a single, terminal sulfur atom. An “alkylthio” group maybe represented as —S— alkyl where alkyl is as defined above. The term“arylthio” is used similarly, and may be represented as —S-aryl, witharyl as defined below. The term “mercapto” refers to —SH.

Aryl groups are cyclic aromatic hydrocarbons that do not containheteroatoms. Aryl groups include monocyclic, bicyclic and polycyclicring systems. Thus, aryl groups include, but are not limited to, phenyl,azulenyl, heptalenyl, biphenylenyl, indacenyl, fluorenyl, phenanthrenyl,triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenyl, anthracenyl,indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments,aryl groups contain 6-14 carbons, and in others from 6 to 12 or even6-10 carbon atoms in the ring portions of the groups. Although thephrase “aryl groups” includes groups containing fused rings, such asfused aromatic-aliphatic ring systems (e.g., indanyl,tetrahydronaphthyl, and the like), it does not include aryl groups thathave other groups, such as alkyl or halo groups, bonded to one of thering members. Rather, groups such as tolyl are referred to assubstituted aryl groups. Representative substituted aryl groups may bemono-substituted or substituted more than once. For example,monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-,5-, or 6-substituted phenyl or naphthyl groups, which may be substitutedwith substituents such as those listed above.

The term “heteroaryl group” refers to a monocyclic or condensed-ringaromatic heterocyclic group containing one or more hetero-atoms selectedfrom O, S and N. If the aromatic heterocyclic group has a condensedring, it can include a partially hydrogenated monocyclic group. Examplesof such a heteroaryl group include a pyrazolyl group, a thiazolyl group,an isothiazolyl group, a thiadiazolyl group, an imidazolyl group, afuryl group, a thienyl group, an oxazolyl group, an isoxazolyl group, apyrrolyl group, an imidazolyl group, a (1,2,3)- and (1,2,4)-triazolylgroup, a tetrazolyl group, a pyranyl group, a pyridyl group, apyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a quinolylgroup, an isoquinolyl group, a benzofuranyl group, an isobenzofuranylgroup, an indolyl group, an isoindolyl group, an indazolyl group, abenzoimidazolyl group, a benzotriazolyl group, a benzoxazolyl group, abenzothiazolyl group, a benzo[b]thiophenyl group, athieno[2,3-b]thiophenyl group, a (1,2)- and (1,3)-benzoxathiol group, achromenyl group, a 2-oxochromenyl group, a benzothiadiazolyl group, aquinolizinyl group, a phthalazinyl group, a naphthyridinyl group, aquinoxalinyl group, a quinazolinyl group, a cinnolinyl group, and acarbazolyl group.

A “derivative” of a compound means a chemically modified compoundwherein the chemical modification takes place at one or more functionalgroups of the compound. The derivative however, is expected to retain,or enhance, the pharmacological activity of the compound from which itis derived.

As used herein, “administering” refers to local and systemicadministration, e.g., including enteral, parenteral, pulmonary, andtopical/transdermal administration. Routes of administration for agents(e.g., hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) that find use in the methods describedherein include, e.g., oral (per os (p.o.)) administration, nasal orinhalation administration, administration as a suppository, topicalcontact, transdermal delivery (e.g., via a transdermal patch),intrathecal (IT) administration, intravenous (“iv”) administration,intraperitoneal (“ip”) administration, intramuscular (“im”)administration, intralesional administration, or subcutaneous (“sc”)administration, or the implantation of a slow-release device e.g., amini-osmotic pump, a depot formulation, etc., to a subject.Administration can be by any route including parenteral and transmucosal(e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteraladministration includes, e.g., intravenous, intramuscular,intra-arterial, intradermal, subcutaneous, intraperitoneal,intraventricular, ionophoretic and intracranial. Other modes of deliveryinclude, but are not limited to, the use of liposomal formulations,intravenous infusion, transdermal patches, etc.

The terms “systemic administration” and “systemically administered”refer to a method of administering the agent(s) described herein orcomposition to a mammal so that the agent(s) or composition is deliveredto sites in the body, including the targeted site of pharmaceuticalaction, via the circulatory system. Systemic administration includes,but is not limited to, oral, intranasal, rectal and parenteral (e.g.,other than through the alimentary tract, such as intramuscular,intravenous, intra-arterial, transdermal and subcutaneous)administration.

The term “co-administering” or “concurrent administration” or“administering in conjunction with” when used, for example with respectto the active agent(s) described herein e.g., hydantoins describedherein, or a tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said hydantoin(s), said stereoisomer(s),or said tautomer(s), or analogues, derivatives, or prodrugs thereof anda second active agent (e.g., a cognition enhancer), refers toadministration of the agent(s) and/the second active agent such thatboth can simultaneously achieve a physiological effect. The two agents,however, need not be administered together. In certain embodiments,administration of one agent can precede administration of the other.Simultaneous physiological effect need not necessarily require presenceof both agents in the circulation at the same time. However, in certainembodiments, co-administering typically results in both agents beingsimultaneously present in the body (e.g., in the plasma) at asignificant fraction (e.g., 20% or greater, preferably 30% or 40% orgreater, more preferably 50% or 60% or greater, most preferably 70% or80% or 90% or greater) of their maximum serum concentration for anygiven dose.

The term “effective amount” or “pharmaceutically effective amount” referto the amount and/or dosage, and/or dosage regime of one or moreagent(s) necessary to bring about the desired result e.g., an amountsufficient to mitigating in a mammal one or more symptoms associatedwith mild cognitive impairment (MCI), or an amount sufficient to lessenthe severity or delay the progression of a disease characterized byamyloid deposits in the brain in a mammal (e.g., therapeuticallyeffective amounts), an amount sufficient to reduce the risk or delayingthe onset, and/or reduce the ultimate severity of a diseasecharacterized by amyloid deposits in the brain in a mammal (e.g.,prophylactically effective amounts).

The phrase “cause to be administered” refers to the actions taken by amedical professional (e.g., a physician), or a person controllingmedical care of a subject, that control and/or permit the administrationof the agent(s) at issue to the subject. Causing to be administered caninvolve diagnosis and/or determination of an appropriate therapeutic orprophylactic regimen, and/or prescribing particular agent(s) for asubject. Such prescribing can include, for example, drafting aprescription form, annotating a medical record, and the like.

As used herein, the terms “treating” and “treatment” refer to delayingthe onset of, retarding or reversing the progress of, reducing theseverity of, or alleviating or preventing either the disease orcondition to which the term applies, or one or more symptoms of suchdisease or condition.

The term “mitigating” refers to reduction or elimination of one or moresymptoms of that pathology or disease, and/or a reduction in the rate ordelay of onset or severity of one or more symptoms of that pathology ordisease, and/or the prevention of that pathology or disease. In certainembodiments, the reduction or elimination of one or more symptoms ofpathology or disease can include, but is not limited to, reduction orelimination of one or more markers that are characteristic of thepathology or disease (e.g., of total-Tau (tTau), phospho-Tau (pTau),APPneo, soluble Aβ40, pTau/Aβ42 ratio and tTau/Aβ42 ratio, and/or anincrease in the CSF of levels of one or more components selected fromthe group consisting of Aβ42/Aβ40 ratio, Aβ42/Aβ38 ratio, sAPPα,sAPPα/sAPPβ ratio, sAPPα/Aβ40 ratio, sAPPα/Aβ42 ratio, etc.) and/orreduction, stabilization or reversal of one or more diagnostic criteria(e.g., clinical dementia rating (CDR)).

As used herein, the phrase “consisting essentially of” refers to thegenera or species of active pharmaceutical agents recited in a method orcomposition, and further can include other agents that, on their own donot substantial activity for the recited indication or purpose. In someembodiments, the phrase “consisting essentially of” expressly excludesthe inclusion of one or more additional agents that haveneuropharmacological activity other than the recited agent(s) (e.g.,other than ASBIs such as galangin, rutin, and analogues, derivatives, orprodrugs thereof). In some embodiments, the phrase “consistingessentially of” expressly excludes the inclusion of one or moreadditional active agents other than the active agent(s) described herein(e.g., other than ASBIs such as galangin, rutin, and analogues,derivatives, or prodrugs thereof). In some embodiments, the phrase“consisting essentially of” expressly excludes the inclusion of one ormore acetylcholinesterase inhibitors.

The terms “subject”, “individual”, and “patient” interchangeably referto a mammal, preferably a human or a non-human primate, but alsodomesticated mammals (e.g., canine or feline), laboratory mammals (e.g.,mouse, rat, rabbit, hamster, guinea pig) and agricultural mammals (e.g.,equine, bovine, porcine, ovine). In various embodiments, the subject canbe a human (e.g., adult male, adult female, adolescent male, adolescentfemale, male child, female child) under the care of a physician or otherhealth worker in a hospital, psychiatric care facility, as anoutpatient, or other clinical context. In certain embodiments thesubject may not be under the care or prescription of a physician orother health worker.

The term “formulation” or “drug formulation” or “dosage form” or“pharmaceutical formulation” as used herein refers to a compositioncontaining at least one therapeutic agent or medication for delivery toa subject. In certain embodiments the dosage form comprises a given“formulation” or “drug formulation” and may be administered to a patientin the form of a lozenge, pill, tablet, capsule, suppository, membrane,strip, liquid, patch, film, gel, spray or other form.

The term “mucosal membrane” refers generally to any of the mucus-coatedbiological membranes in the body. In certain embodiments active agent(s)described herein can be administered herein via any mucous membranefound in the body, including, but not limited to buccal, perlingual,nasal, sublingual, pulmonary, rectal, and vaginal mucosa. Absorptionthrough the mucosal membranes of the oral cavity and those of the gutare of interest. Thus, peroral, buccal, sublingual, gingival and palatalabsorption are contemplated herein.

The term “transmucosal” delivery of a drug and the like is meant toencompass all forms of delivery across or through a mucosal membrane.

The term “bioadhesion” as used herein refers to the process of adhesionof the dosage form(s) to a biological surface, e.g., mucosal membranes.

“Controlled drug delivery” refers to release or administration of a drugfrom a given dosage form in a controlled fashion in order to achieve thedesired pharmacokinetic profile in vivo. An aspect of“controlled” drugdelivery is the ability to manipulate the formulation and/or dosage formin order to establish the desired kinetics of drug release.

“Sustained drug delivery” refers to release or administration of a drugfrom a source (e.g., a drug formulation) in a sustained fashion over aprotracted yet specific period of time, that may extend from severalminutes to a few hours, days, weeks or months. In various embodimentsthe term “sustained” will be used to refer to delivery of consistentand/oe substantially constant levels of drug over a time period rangingfrom a few minutes to a day, with a profile characterized by the absenceof an immediate release phase, such as the one obtained from IVadministration.

The term “T_(max)” as used herein means the time point of maximumobserved plasma concentration.

The term “C_(max)” as used herein means the maximum observed plasmaconcentration.

The term “plasma t_(1/2)” as used herein means the observed “plasmahalf-life” and represents the time required for the drug plasmaconcentration to reach the 50% of its maximal value (C_(max)). Thisfacilitates determination of the mean duration of pharmacologicaleffects. In addition, it facilitates direct and meaningful comparisonsof the duration of different test articles after delivery via the sameor different routes.

The term “Optimal Therapeutic Targeting Ratio” or “OTTR” represents theaverage time that the drug is present at therapeutic levels, defined astime within which the drug plasma concentration is maintained above 50%of C_(max) normalized by the drug's elimination half-life multiplied bythe ratio of the C_(max) obtained in the dosage form of interest overthe C_(max) following IV administration of equivalent doses and it iscalculated by the formula:

OTTR=(C ^(IV) _(max) /C _(max))×(Dose/Dose^(IV))(Time above 50% of C_(max))/(Terminal^(IV) elimination half-life of the drug).

The term “substantiall pure” means sufficiently homogeneous to appearfree of readily detectable impurities as determined by standard methodsof analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicalor chemical properties, of the compound. Methods for purification of thecompounds to produce substantially chemically pure compounds are knownto those of skill in the art. A substantially chemically pure compoundmay, however, be a mixture of stereoisomers or isomers. In suchinstances, further purification might increase the specific activity ofthe compound.

The term “substantially pure” when used with respect to enantiomersindicates that one particular enantiomer (e.g. an S enantiomer or an Renantiomer) is substantially free of its stereoisomer. In variousembodiments substantially pure indicates that a particular enantiomer isat least 70%, or at least 80%, or at least 90%, or at least 95%, or atleast 98%, or at least 99% of the purified compound. Methods ofproducing substantially pure enantiomers are well known to those ofskill in the art. For example, a single stereoisomer, e.g., anenantiomer, substantially free of its stereoisomer may be obtained byresolution of the racemic mixture using a method such as formation ofdiastereomers using optically active resolving agents (Stereochemistryof Carbon Compounds, (1962) by E. L. Eliel, McGraw Hill; Lochmuller(1975) J. Chromatogr., 113(3): 283-302). Racemic mixtures of chiralcompounds of the can be separated and isolated by any suitable method,including, but not limited to: (1) formation of ionic, diastereomericsalts with chiral compounds and separation by fractional crystallizationor other methods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. Anotherapproach for separation of the enantiomers is to use a Diacel chiralcolumn and elution using an organic mobile phase such as done by ChiralTechnologies (www.chiraltech.com) on a fee for service basis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates various hydantoins.

FIG. 2 illustrates various hydantoins.

FIG. 3 models of proposed interaction of the hydantoin with the FLAPregion of BACE1. The lower panel illustrates interaction of the B-ring3,4-substituent with the FLAP, Trp76 disrupts Trp-76-→Tyr-71 H-bondingcausing Tyr-71 to flip to the left and interact with the difluorocontaining A ring.

FIG. 4 illustrates APP Binding BACE Inhibitor (ABBI) FAH-3 binding toeAPP₅₇₅₋₆₂₄ as measured by surface plasmon resonance (SPR) screening.The binding affinity of the compounds for the ectodomain of APP wasdetermined using SPR. We have developed a technique for measuring theaffinity of compounds to fragments of the ectodomain of APP. For thecompound 3 binding experiments a TRX-eAPP575-624 substrate was used. TheeAPP was crosslinked linked to the CM5 Biacore chips (GE Healthcare).Compound 3 at various concentrations were used in the flow through overthe chip and the plasmon resonance signal was determined using a BiacoreT100.

FIG. 5 illustrates inhibition of Aβ production by FAH-3.

FIG. 6A illustrates selectivity of ABBI for inhibition of the APP-BACEcleavage as compared to the PSGL-BACE cleavage shown in FIG. 6B.

DETAILED DESCRIPTION

In various embodiments, hydantoins are identified that appear to inhibitβ-secretase mediated APP processing by a novel mechanism. In particular,without being bound to a particular theory, it is believed that thesemolecules interact with BACE and/or with APP and/or with a BACE/APPcomplex and thereby inhibit the BACE cleavage of the MBP-C125 APPsubstrate, resulting in the inhibition of the production of C99 and theβ-site peptide substrate (P5-P5′). In addition, the various hydantoinsidentified herein inhibit Aβ42 in neuroblastoma SHSY5Y cells. Further wedemonstrate the activity of the hydantoins identified herein appears tobe associated with binding to BACE and/or to APP particularly when thesemoieties form a BACE/APP complex. Accordingly, it is believed thecompounds described herein represent a new class of compounds designatedherein as APP-Binding-BACE Inhibitors (ABBIs) and provide a newmechanism to modulate APP processing. The hydantoins described hereinappear to show improved brain permeability and functional BACEinhibition.

The ABBIs are specific for the APP and/or BACE and/or the APP/BACEcomplex and are believed to show fewer undesired side-effects becausethe ABBIs are typically not active on other substrates for the enzyme orother enzyme complexes. With respect to inhibitors of γ-secretase,substrates other than APP, such as Notch, raise concerns for potentialside effects of γ-secretase inhibition, and the recent failure of theγ-secretase inhibitor, Semagacestat, serves to reinforce such concerns.Similarly in the case of BACE, for example, inhibition of non-APPsubstrates such as PSGL1 or LRP could produce adverse side-effects.Therefore, a desirable BACE inhibitor would be one that wouldbind/interact not with BACE but rather to APP, or to the APP/BACEcomplex leading to APP-specific BACE complex inhibition (ABBI).

Such ABBIs would potentially interact with the APP-BACE complex, e.g.,at the membrane and prevent its transition to the “active” complex inearly endosomes, where at pH<5 BACE is fully active. Some β-site bindingantibodies have been shown to block the cleavage of APP by BACE and alsowork in animal models of AD, however for effective pharmaceuticaldevelopment small organic molecules are typically preferred torelatively large biomolecules such as antibodies.

The data we report herein on the identification of the first ABBIsdemonstrates that such an approach is feasible. Without being bound to aparticular theory, ABBIs appear to inhibit BACE activity by interactingwith APP, particular when in an APP/BACE complex thereby inhibiting theBACE cleavage of the Amyloid Precursor Protein (APP) but not theproteolytic cleavage of other substrates. Such therapeutics are believedto represent a new class of Alzheimer's disease (or other amyloidogenicdisease) therapeutics.

The active site of BACE1 is covered by flaps. A single flap of 14residues in length forms an α-hairpin structure that is perpendicular toa cleft that houses the active site and covers the central part of thatactive site. During the catalytic cycle, the flaps open to allowentrance of substrate (APP) into the catalytic cleft and also to releasehydrolytic products. Initially, hydantoins described herein wereproduced by introducing a dihalo (e.g., difluoro) ring into the aminohydantoin of Compound 0 (shown in FIG. 1) to produce compound 1 (alsoshown in FIG. 1). Without being bound to a particular theory, it isbelieved the dihalo-ring introduces a FLAP interaction (e.g. aninteraction with FLAP residuces Tyr-71 (through pi-stacking) and Trp-76(through interaction with OCF2)) and restricts FLAP movement limitingAPP entry into the active site and/or the exit of cleavage products(see, e.g., FIG. 3). This provides a new family of small (MW<400) brainpenetrant BACE inhibitors (ABCIs).

Compound 2 and other hydantoins were produced (see, e.g., compounds 1-5pharmacokinetic evaluation of these hydantoins was determined in brainuptake assays using NTg mice (see, e.g., Table 1). It was alsodetermined that Compound 1 lowered Aβ42 in the same animals at 5 mpk,while compound 3 lowered Aβ at 1 mpk.

TABLE 1 Biological properties of illustrative hydantoins as compared toBACE IV (β-sectretase inhibitor IV from Calbiochem (cat #565788). BACEAPP Brain/Plasma IC₅₀ binding Ratio Compound (μM) Kd (uM) at Cmax MWFAH-1 3 5 ~3:1 301.3 (moderate binding) FAH-2 2 8 ~1:1 367.3 (moderatebinding) FAH-3 0.52 0.3 ~1:2 381.3 (strong binding) FAH-4 2.1 ~3:1 367.3FAH-5 5.0 329.3  FAH-17 0.15 <1 uM ~5:1 363.3 (strong binding) *BACE0.05 >50 <0.1:1   578 inhibitor (essentially IV no binding)*β-sectretase inhibitor IV from Calbiochem (cat #565788)

It was also demonstrated that the compounds interacted with eAPP (see,e.g., FIG. 4) using a BiaCore assay. The hydantoins contemplated hereinthus show desirable pharmacokinetic profiles and have the desiredactivity as evidenced by interaction with APP and/or BACE/APP complexesand lowering of Aβ42.

The sequential cleavage of APP by membrane-bound proteases β-secretaseand γ-secretase results in the formation of Aβ. The β-Site APP cleavageenzyme-1 (BACE1) was identified as the major β-secretase activity thatmediates the first cleavage of APP in the (β-amyloidogenic pathway. Inview of the ability of the ABBI compounds described herein tospecifically block BACE1 activity at APP, it is believed (and the datapresented herein show) that these ABBI compounds can lower Aβ levels orprevent the formation of the neurotoxic Aβ species. Accordingly, thesecompounds are believed to prevent or slow the progression of the diseaseand/or to prevent or slow the progression of pre-clinical manifestationsof the amyloidogenic disease pathway.

Accordingly it is believed that these agents) (e.g., hydantoinsdescribed herein, or a tautomer(s) or stereoisomer(s) thereof, orpharmaceutically acceptable salts or solvates of said hydantoin(s), saidstereoisomer(s), or said tautomer(s), or analogues, derivatives, orprodrugs thereof) can be used to prevent or delay the onset of apre-Alzheimer's cognitive dysfunction, and/or to ameliorate one or moresymptoms of a pre-Alzheimer's cognitive dysfunction, and/or to preventor delay the progression of a pre-Alzheimer's condition or cognitivedysfunction to Alzheimer's disease, and/or to promote the processing ofamyloid precursor protein (APP) by the non-amyloidogenic pathway. Incertain embodiments these agents can be used in the treatment ofAlzheimer's disease (e.g., to lessen the severity of the disease, and/orto ameliorate one or more symptoms of the disease, and/or to slow theprogression of the disease).

Therapeutic and Prophylactic Methods.

In various embodiments therapeutic and/or prophylactic methods areprovided that utilize the active agent(s) (e.g., hydantoins describedherein, or a tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said hydantoin(s), said stereoisomer(s),or said tautomer(s), or analogues, derivatives, or prodrugs thereof) areprovided. Typically the methods involve administering one or more activeagent(s) to a subject (e.g., to a human in need thereof) in an amountsufficient to realize the desired therapeutic or prophylactic result.

Prophylaxis

In certain embodiments active agent(s) (e.g., hydantoins describedherein, or a tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said hydantoin(s), said stereoisomer(s),or said tautomer(s), or analogues, derivatives, or prodrugs thereof) areutilized in various prophylactic contexts. Thus, for example, in certainembodiments, the active agent(s) can be used to prevent or delay theonset of a pre-Alzheimer's cognitive dysfunction, and/or to ameliorateone more symptoms of a pre-Alzheimer's condition and/or cognitivedysfunction, and/or to prevent or delay the progression of apre-Alzheimer's condition and/or cognitive dysfunction to Alzheimer'sdisease.

Accordingly in certain embodiments, the prophylactic methods describedherein are contemplated for subjects identified as “at risk” and/or ashaving evidence of early Alzheimer's Disease (AD) pathological changes,but who do not meet clinical criteria for MCI or dementia. Without beingbound to a particular theory, it is believed that even this“preclinical” stage of the disease represents a continuum fromcompletely asymptomatic individuals with biomarker evidence suggestiveof AD-pathophysiological process(es) (abbreviated as AD-P, see, e.g.,Sperling et al. (2011) Alzheimer's & Dementia, 1-13) at risk forprogression to AD dementia to biomarker-positive individuals who arealready demonstrating very subtle decline but not yet meetingstandardized criteria for MCI (see, e.g., Albert et al. (2011)Alzheimer's and Dementia, 1-10 (doi:10.1016/j.jalz.2011.03.008).

This latter group of individuals might be classified as “not normal, notMCI” but would be can be designated “pre-symptomatic” or “pre-clinicalor “asymptomatic” or “premanifest”). In various embodiments thiscontinuum of pre-symptomatic AD can also encompass, but is notnecessarily limited to, (1) individuals who carry one or moreapolipoprotein E (APOE) ε4 alleles who are known or believed to have anincreased risk of developing AD dementia, at the point they are AD-Pbiomarker-positive, and (2) carriers of autosomal dominant mutations,who are in the presymptomatic biomarker-positive stage of their illness,and who will almost certainly manifest clinical symptoms and progress todementia.

A biomarker model has been proposed in which the most widely validatedbiomarkers of AD-P become abnormal and likewise reach a ceiling in anordered manner (see, e.g., Jack et al. (2010) Lancet Neurol., 9:119-128.). This biomarker model parallels proposed pathophysiologicalsequence of (pre-AD/AD), and is relevant to tracking the preclinical(asymptomatic) stages of AD (see, e.g., FIG. 3 in Sperling et al. (2011)Alzheimer's & Dementia, 1-13). Biomarkers of brain amyloidosis include,but are not limited to reductions in CSF Aβ₄₂ and increased amyloidtracer retention on positron emission tomography (PET) imaging. ElevatedCSF tau is not specific to AD and is thought to be a biomarker ofneuronal injury. Decreased fluorodeoxyglucose 18F (FDG) uptake on PETwith a temporoparietal pattern of hypometabolism is a biomarker ofAD-related synaptic dysfunction. Brain atrophy on structural magneticresonance imaging (MRI) in a characteristic pattern involving the medialtemporal lobes, paralimbic and temporoparietal cortices is a biomarkerof AD-related neurodegeneration. Other markers include, but are notlimited to volumetric MRI, FDG-PET, or plasma biomarkers (see, e.g.,Vemuri et al. (2009) Neurology, 73: 294-301; Yaffe et al. (2011) JAMA305: 261-266).

In certain embodiments the subjects suitable for the prophylacticmethods contemplated herein include, but are not limited to, subjectscharacterized as having asymptomatic cerebral amyloidosis. In variousembodiments these individuals have biomarker evidence of Aβ accumulationwith elevated tracer retention on PET amyloid imaging and/or low Aβ42 inCSF assay, but typically no detectable evidence of additional brainalterations suggestive of neurodegeneration or subtle cognitive and/orbehavioral symptomatology.

It is noted that currently available CSF and PET imaging biomarkers ofAβ primarily provide evidence of amyloid accumulation and deposition offibrillar forms of amyloid. Data suggest that soluble or oligomericforms of Aβ are likely in equilibrium with plaques, which may serve asreservoirs. In certain embodiments it is contemplated that there is anidentifiable preplaque stage in which only soluble forms of Aβ arepresent. In certain embodiments it is contemplated that oligomeric formsof amyloid may be critical in the pathological cascade, and provideuseful markers. In addition, early synaptic changes may be presentbefore evidence of amyloid accumulation.

In certain embodiments the subjects suitable for the prophylacticmethods contemplated herein include, but are not limited to, subjectscharacterized as amyloid positive with evidence of synaptic dysfunctionand/or early neurodegeneration. In various embodiments these subjectshave evidence of amyloid positivity and presence of one or more markersof “downstream” AD-related neuronal injury. Illustrative, butnon-limiting markers of neuronal injury include, but are not limited to(1) elevated CSF tau or phospho-tau, (2) hypometabolism in an AD-likepattern (i.e., posterior cingulate, precuneus, and/or temporoparietalcortices) on FDG-PET, and (3) cortical thinning/gray matter loss in aspecific anatomic distribution (i.e., lateral and medial parietal,posterior cingulate, and lateral temporal cortices) and/or hippocampalatrophy on volumetric MRI. Other markers include, but are not limited tofMRI measures of default network connectivity. In certain embodimentsearly synaptic dysfunction, as assessed by functional imaging techniquessuch as FDG-PET and fMRI, can be detectable before volumetric loss.Without being bound to a particular theory, it is believed thatamyloid-positive individuals with evidence of early neurodegenerationmay be farther down the trajectory (i.e., in later stages of preclinical(asymptomatic) AD).

In certain embodiments the subjects suitable for the prophylacticmethods contemplated herein include, but are not limited to, subjectscharacterized as amyloid positive with evidence of neurodegeneration andsubtle cognitive decline. Without being bound to a particular theory, itis believed that those individuals with biomarker evidence of amyloidaccumulation, early neurodegeneration, and evidence of subtle cognitivedecline are in the last stage of preclinical (asymptomatic) AD, and areapproaching the border zone with clinical criteria for mild cognitiveimpairment (MCI). These individuals may demonstrate evidence of declinefrom their own baseline (particularly if proxies of cognitive reserveare taken into consideration), even if they still perform within the“normal” range on standard cognitive measures. Without being bound to aparticular theory, it is believed that more sensitive cognitivemeasures, particularly with challenging episodic memory measures, maydetect very subtle cognitive impairment in amyloid-positive individuals.In certain embodiments criteria include, but are not limited to,self-complaint of memory decline or other subtle neurobehavioralchanges.

As indicated above, subjects/patients amenable to prophylactic methodsdescribed herein include individuals at risk of disease (e.g., apathology characterized by amyloid plaque formation such as MCI) but notshowing symptoms, as well as subjects presently showing certain symptomsor markers. It is known that the risk of MCI and later Alzheimer'sdisease generally increases with age. Accordingly, in asymptomaticsubjects with no other known risk factors, in certain embodiments,prophylactic application is contemplated for subjects over 50 years ofage, or subjects over 55 years of age, or subjects over 60 years of age,or subjects over 65 years of age, or subjects over 70 years of age, orsubjects over 75 years of age, or subjects over 80 years of age, inparticular to prevent or slow the onset or ultimate severity of mildcognitive impairment (MCI), and/or to slow or prevent the progressionfrom MCI to early stage Alzheimer's disease (AD).

In certain embodiments, the methods described herein are especiallyuseful for individuals who do have a known genetic risk of Alzheimer'sdisease (or other amyloidogenic pathologies), whether they areasymptomatic or showing symptoms of disease. Such individuals includethose having relatives who have experienced MCI or AD (e.g., a parent, agrandparent, a sibling), and those whose risk is determined by analysisof genetic or biochemical markers. Genetic markers of risk towardAlzheimer's disease include, for example, mutations in the APP gene,particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively (see Hardy(1997) Trends. Neurosci., 20: 154-159). Other markers of risk includemutations in the presenilin genes (PS1 and PS2), family history of AD,having the familial Alzheimer's disease (FAD) mutation, the APOE ε4allele, hypercholesterolemia or atherosclerosis. Further susceptibilitygenes for the development of Alzheimer's disease are reviewed, e.g., inSleegers, et al. (2010) Trends Genet. 26(2): 84-93.

In some embodiments, the subject is asymptomatic but has familial and/orgenetic risk factors for developing MCI or Alzheimer's disease. Inasymptomatic patients, treatment can begin at any age (e.g., at about20, about 30, about 40, about 50 years of age). Usually, however, it isnot necessary to begin treatment until a patient reaches at least about40, or at least about 50, or at least about 55, or at least about 60, orat least about 65, or at least about 70 years of age.

In some embodiments, the subject exhibits symptoms, for example, of mildcognitive impairment (MCI) or Alzheimer's disease (AD). Individualspresently suffering from Alzheimer's disease can be recognized fromcharacteristic dementia, as well as the presence of risk factorsdescribed above. In addition, a number of diagnostic tests are availablefor identifying individuals who have AD. These include measurement ofCSF Tau, phospho-tau (pTau), Aβ42 levels and C-terminally cleaved APPfragment (APPneo). Elevated total-Tau (tTau), phospho-Tau (pTau),APPneo, soluble Aβ40, pTau/Aβ42 ratio and tTau/Aβ42 ratio, and decreasedAβ42 levels, Aβ42/Aβ40 ratio, Aβ42/Aβ38 ratio, sAPPα levels, sAPPα/sAPPβratio, sAPPα/Aβ40 ratio, and sAPPα/Aβ42 ratio signify the presence ofAD. In some embodiments, the subject or patient is diagnosed as havingMCI. Increased levels of neural thread protein (NTP) in urine and/orincreased levels of α2-macroglobulin (α2M) and/or complement factor H(CFH) in plasma are also biomarkers of MCI and/or AD (see, e.g., Anoopet al. (2010) Int. J. Alzheimer's Dis. 2010:606802).

In certain embodiments, subjects amenable to treatment may haveage-associated memory impairment (AAMI), or mild cognitive impairment(MCI). The methods described herein are particularly well-suited to theprophylaxis and/or treatment of MCI. In such instances, the methods candelay or prevent the onset of MCI, and or reduce one or more symptomscharacteristic of MCI and/or delay or prevent the progression from MCIto early-, mid- or late-stage Alzheimer's disease or reduce the ultimateseverity of the disease.

Mild Cognitive Impairment (MCI)

Mild cognitive impairment (MCI, also known as incipient dementia, orisolated memory impairment) is a diagnosis given to individuals who havecognitive impairments beyond that expected for their age and education,but that typically do not interfere significantly with their dailyactivities (see, e.g., Petersen et a. (1999) Arch. Neurol. 56(3):303-308). It is considered in many instances to be a boundary ortransitional stage between normal aging and dementia. Although MCI canpresent with a variety of symptoms, when memory loss is the predominantsymptom it is termed “amnestic MCI” and is frequently seen as a riskfactor for Alzheimer's disease (see, e.g., Grundman et al. (2004) Arch.Neurol. 61(1): 59-66; and on the internet aten.wikipedia.org/wiki/Mild_cognitive_impairment-cite_note-Grundman-1).When individuals have impairments in domains other than memory it isoften classified as non-amnestic single- or multiple-domain MCI andthese individuals are believed to be more likely to convert to otherdementias (e.g., dementia with Lewy bodies). There is evidencesuggesting that while amnestic MCI patients may not meet neuropathologiccriteria for Alzheimer's disease, patients may be in a transitionalstage of evolving Alzheimer's disease; patients in this hypothesizedtransitional stage demonstrated diffuse amyloid in the neocortex andfrequent neurofibrillary tangles in the medial temporal lobe (see, e.g.,Petersen et al. (2006) Arch. Neurol. 63(5): 665-72).

The diagnosis of MCI typically involves a comprehensive clinicalassessment including clinical observation, neuroimaging, blood tests andneuropsychological testing. In certain embodiments diagnostic criteriafor MIC include, but are not limited to those described by Albert et al.(2011) Alzheimer's & Dementia. 1-10. As described therein, diagnosticcriteria include (1) core clinical criteria that could be used byhealthcare providers without access to advanced imaging techniques orcerebrospinal fluid analysis, and (2) research criteria that could beused in clinical research settings, including clinical trials. Thesecond set of criteria incorporate the use of biomarkers based onimaging and cerebrospinal fluid measures. The final set of criteria formild cognitive impairment due to AD has four levels of certainty,depending on the presence and nature of the biomarker findings.

In certain embodiments clinical evaluation/diagnosis of MCI involves:(1) Concern reflecting a change in cognition reported by patient orinformant or clinician (i.e., historical or observed evidence of declineover time); (2) Objective evidence of Impairment in one or morecognitive domains, typically including memory (i.e., formal or bedsidetesting to establish level of cognitive function in multiple domains);(3) Preservation of independence in functional abilities; (4) Notdemented; and in certain embodiments, (5) An etiology of MCI consistentwith AD pathophysiological processes. Typically vascular, traumatic, andmedical causes of cognitive decline, are ruled out where possible. Incertain embodiments, when feasible, evidence of longitudinal decline incognition is identified. Diagnosis is reinforced by a history consistentwith AD genetic factors, where relevant.

With respect to impairment in cognitive domain(s), there should beevidence of concern about a change in cognition, in comparison with theperson's previous level. There should be evidence of lower performancein one or more cognitive domains that is greater than would be expectedfor the patient's age and educational background. If repeatedassessments are available, then a decline in performance should beevident over time. This change can occur in a variety of cognitivedomains, including memory, executive function, attention, language, andvisuospatial skills. An impairment in episodic memory (i.e., the abilityto learn and retain new information) is seen most commonly in MCIpatients who subsequently progress to a diagnosis of AD dementia.

With respect to preservation of independence in functional abilities, itis noted that persons with MCI commonly have mild problems performingcomplex functional tasks which they used to perform shopping. They maytake more time, be less efficient, and make more errors at performingsuch activities than in the past. Nevertheless, they generally maintaintheir independence of function in daily life, with minimal aids orassistance.

With respect to dementia, the cognitive changes should be sufficientlymild that there is no evidence of a significant impairment in social oroccupational functioning. If an individual has only been evaluated once,change will be inferred from the history and/or evidence that cognitiveperformance is impaired beyond what would have been expected for thatindividual.

Cognitive testing is optimal for objectively assessing the degree ofcognitive impairment for an individual. Scores on cognitive tests forindividuals with MCI are typically 1 to 1.5 standard deviations belowthe mean for their age and education matched peers on culturallyappropriate normative data (i.e., for the impaired domain(s), whenavailable).

Episodic memory (i.e., the ability to learn and retain new information)is most commonly seen in MCI patients who subsequently progress to adiagnosis of AD dementia. There are a variety of episodic memory teststhat are useful for identifying those MCI patients who have a highlikelihood of progressing to AD dementia within a few years. These teststypically assess both immediate and delayed recall, so that it ispossible to determine retention over a delay. Many, although not all, ofthe tests that have proven useful in this regard are wordlist learningtests with multiple trials. Such tests reveal the rate of learning overtime, as well as the maximum amount acquired over the course of thelearning trials. They are also useful for demonstrating that theindividual is, in fact, paying attention to the task on immediaterecall, which then can be used as a baseline to assess the relativeamount of material retained on delayed recall. Examples of such testsinclude (but are not limited to: the Free and Cued Selective RemindingTest, the Rey Auditory Verbal Learning Test, and the California VerbalLearning Test. Other episodic memory measures include, but are notlimited to: immediate and delayed recall of a paragraph such as theLogical Memory I and II of the Wechsler Memory Scale Revised (or otherversions) and immediate and delayed recall of nonverbal materials, suchas the Visual Reproduction subtests of the Wechsler Memory Scale-RevisedI and II.

Because other cognitive domains can be impaired among individuals withMCI, it is desirable to examine domains in addition to memory. Theseinclude, but are not limited to executive functions (e.g., set-shifting,reasoning, problem-solving, planning), language (e.g., naming, fluency,expressive speech, and comprehension), visuospatial skills, andattentional control (e.g., simple and divided attention). Many clinicalneuropsychological measures are available to assess these cognitivedomains, including (but not limited to the Trail Making Test (executivefunction), the Boston Naming Test, letter and category fluency(language), figure copying (spatial skills), and digit span forward(attention).

As indicated above, genetic factors can be incorporated into thediagnosis of MCI. If an autosomal dominant form of AD is known to bepresent (i.e., mutation in APP, PS1, PS2), then the development of MCIis most likely the precursor to AD dementia. The large majority of thesecases develop early onset AD (i.e., onset below 65 years of age).

In addition, there are genetic influences on the development of lateonset AD dementia. For example, the presence of one or two ε4 alleles inthe apolipoprotein E (APOE) gene is a genetic variant broadly acceptedas increasing risk for late-onset AD dementia. Evidence suggests that anindividual who meets the clinical, cognitive, and etiologic criteria forMCI, and is also APOE ε4 positive, is more likely to progress to ADdementia within a few years than an individual without this geneticcharacteristic. It is believed that additional genes play an important,but smaller role than APOE and also confer changes in risk forprogression to AD dementia (see, e.g., Bertram et al. (2010) Neuron, 21:270-281).

In certain embodiments subjects suitable for the prophylactic methodsdescribed herein include, but need not be limited to, subjectsidentified having one or more of the core clinical criteria describedabove and/or subjects identified with one or more “research criteria”for MCI, e.g., as described below.

“Research criteria” for the identification/prognosis of MCI include, butare not limited to biomarkers that increase the likelihood that MCIsyndrome is due to the pathophysiological processes of AD. Without beingbound to a particular theory, it is believed that the conjointapplication of clinical criteria and biomarkers can result in variouslevels of certainty that the MCI syndrome is due to ADpathophysiological processes. In certain embodiments, two categories ofbiomarkers have been the most studied and applied to clinical outcomesare contemplated. These include “AP” (which includes CSF Aβ₄₂ and/or PETamyloid imaging) and “biomarkers of neuronal injury” (which include, butare not limited to CSF tau/p-tau, hippocampal, or medial temporal lobeatrophy on MRI, and temporoparietal/precuneus hypometabolism orhypoperfusion on PET or SPECT).

Without being bound to a particular theory, it is believed that evidenceof both Aβ, and neuronal injury (either an increase in tau/p-tau orimaging biomarkers in a topographical pattern characteristic of AD),together confers the highest probability that the AD pathophysiologicalprocess is present. Conversely, if these biomarkers are negative, thismay provide information concerning the likelihood of an alternatediagnosis. It is recognized that biomarker findings may be contradictoryand accordingly any biomarker combination is indicative (an indicator)used on the context of a differential diagnosis and not itselfdispositive. It is recognized that varying severities of an abnormalitymay confer different likelihoods or prognoses, that are difficult toquantify accurately for broad application.

For those potential MCI subjects whose clinical and cognitive MCIsyndrome is consistent with AD as the etiology, the addition ofbiomarker analysis effects levels of certainty in the diagnosis. In themost typical example in which the clinical and cognitive syndrome of MCIhas been established, including evidence of an episodic memory disorderand a presumed degenerative etiology, the most likely cause is theneurodegenerative process of AD. However, the eventual outcome still hasvariable degrees of certainty. The likelihood of progression to ADdementia will vary with the severity of the cognitive decline and thenature of the evidence suggesting that AD pathophysiology is theunderlying cause. Without being bound to a particular theory it isbelieved that positive biomarkers reflecting neuronal injury increasethe likelihood that progression to dementia will occur within a fewyears and that positive findings reflecting both Aβ accumulation andneuronal injury together confer the highest likelihood that thediagnosis is MCI due to AD.

A positive Aβ biomarker and a positive biomarker of neuronal injuryprovide an indication that the MCI syndrome is due to AD processes andthe subject is well suited for the methods described herein.

A positive Aβ biomarker in a situation in which neuronal injurybiomarkers have not been or cannot be tested or a positive biomarker ofneuronal injury in a situation in which Aβ biomarkers have not been orcannot be tested indicate an intermediate likelihood that the MCIsyndrome is due to AD. Such subjects are believed to be is well suitedfor the methods described herein

Negative biomarkers for both Aβ and neuronal injury suggest that the MCIsyndrome is not due to AD. In such instances the subjects may not bewell suited for the methods described herein.

There is evidence that magnetic resonance imaging can observedeterioration, including progressive loss of gray matter in the brain,from mild cognitive impairment to full-blown Alzheimer disease (see,e.g., Whitwell et al. (2008) Neurology 70(7): 512-520). A techniqueknown as PiB PET imaging is used to clearly show the sites and shapes ofbeta amyloid deposits in living subjects using a C11 tracer that bindsselectively to such deposits (see, e.g., Jack et al. (2008) Brain 131(Pt 3): 665-680).

In certain embodiments, MCI is typically diagnosed when there is 1)Evidence of memory impairment; 2) Preservation of general cognitive andfunctional abilities; and 3) Absence of diagnosed dementia.

In certain embodiments MCI and stages of Alzheimer's disease can beidentified/categorized, in part by Clinical Dementia Rating (CDR)scores. The CDR is a five point scale used to characterize six domainsof cognitive and functional performance applicable to Alzheimer diseaseand related dementias: Memory, Orientation, Judgment & Problem Solving,Community Affairs, Home & Hobbies, and Personal Care. The information tomake each rating can be obtained through a semi-structured interview ofthe patient and a reliable informant or collateral source (e.g., familymember).

The CDR table provides descriptive anchors that guide the clinician inmaking appropriate ratings based on interview data and clinicaljudgment. In addition to ratings for each domain, an overall CDR scoremay be calculated through the use of an algorithm. This score is usefulfor characterizing and tracking a patient's level ofimpairment/dementia: 0=Normal; 0.5=Very Mild Dementia; 1=Mild Dementia;2=Moderate Dementia; and 3=Severe Dementia. An illustrative CDR table isshown in Table 2.

TABLE 2 Illustrative clinical dementia rating (CDR) table. Impairment:None Questionable Mild Moderate Severe CDR: 0 0.5 1 2 3 Memory No memoryConsistent Moderate Severe Severe loss or slight slight memory loss;memory memory inconsistent forgetfulness; more marked loss; only loss;only forgetfulness partial for recent highly fragments recollectionevents; defect learned remain of events' interferes material “benign”with retained; forgetfulness everyday new material activities rapidlylost Orientation Fully Fully Moderate Severe Oriented to orientedoriented difficulty difficulty person only except for with time withtime slight relationships; relationships; difficulty oriented forusually with time place at disoriented relationships examination; totime, often may have to place. geographic disorientation elsewhereJudgment & Solves Slight Moderate Severely Unable to Problem everydayimpairment difficulty in impaired in make Solving problems & in solvinghandling handling judgments handles problems, problems, problems, orsolve business & similarities, similarities similarities problemsfinancial and and and affairs well; differences differences;differences; judgment social social good in judgment judgment relationto usually usually past maintained impaired performance CommunityIndependent Slight Unable to No pretense of independent Affairs functionat impairment function function outside of home usual level in theseindependently Appears well Appears too in job, activities at theseenough to be ill to be shopping, activities taken to taken to volunteer,although may functions functions and social still be outside a outside agroups engaged in family home family some; home. appears normal tocasual inspection Home and Life at Life at home, Mild bit Only simple NoHobbies home, hobbies, and definite chores significant hobbies, andintellectual impairment preserved; function in intellectual interests offunction at very home interests slightly home; more restricted wellimpaired difficult interests, maintained chores poorly abandoned;maintained more complicated hobbies and interests abandoned PersonalFully capable of self-care Needs Requires Requires Care promptingassistance in much help dressing, with hygiene, personal keeping ofcare; personal frequent effects incontinence

A CDR rating of ˜0.5 or ˜0.5 to 1.0 is often considered clinicallyrelevant MCI. Higher CDR ratings can be indicative of progression intoAlzheimer's disease.

In certain embodiments administration of one or more agents describedherein (e.g., hydantoins described herein, or a tautomer(s) orstereoisomer(s) thereof, or pharmaceutically acceptable salts orsolvates of said hydantoin(s), said stereoisomer(s), or saidtautomer(s), or analogues, derivatives, or prodrugs thereof) is deemedeffective when there is a reduction in the CSF of levels of one or morecomponents selected from the group consisting of Tau, phospho-Tau(pTau), APPneo, soluble Aβ40, soluble Aβ42, and/or Aβ42/Aβ40 ratio,and/or when there is a reduction of the plaque load in the brain of thesubject, and/or when there is a reduction in the rate of plaqueformation in the brain of the subject, and/or when there is animprovement in the cognitive abilities of the subject, and/or when thereis a perceived improvement in quality of life by the subject, and/orwhen there is a significant reduction in clinical dementia rating (CDR),and/or when the rate of increase in clinical dementia rating is slowedor stopped and/or when the progression from MCI to early stage AD isslowed or stopped.

In some embodiments, a diagnosis of MCI can be determined by consideringthe results of several clinical tests. For example, Grundman, et al.(2004) Arch Neurol 61: 59-66, report that a diagnosis of MCI can beestablished with clinical efficiency using a simple memory test(paragraph recall) to establish an objective memory deficit, a measureof general cognition (Mini-Mental State Exam (MMSE), discussed ingreater detail below) to exclude a broader cognitive decline beyondmemory, and a structured clinical interview (CDR) with patients andcaregivers to verify the patient's memory complaint and memory loss andto ensure that the patient was not demented. Patients with MCI perform,on average, less than 1 standard deviation (SD) below normal onnonmemorycognitive measures included in the battery. Tests of learning,attention, perceptual speed, category fluency, and executive functionmay be impaired in patients with MCI, but these are far less prominentthan the memory deficit.

Alzheimer's Disease (AD).

In certain embodiments the active agent(s (e.g., hydantoins describedherein, or a tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said hydantoin(s), said stereoisomer(s),or said tautomer(s), or analogues, derivatives, or prodrugs thereof) arecontemplated for the treatment of Alzheimer's disease. In such instancesthe methods described herein are useful in preventing or slowing theonset of Alzheimer's disease (AD), in reducing the severity of AD whenthe subject has transitioned to clinical AD diagnosis, and/or inmitigating one or more symptoms of Alzheimer's disease.

In particular, where the Alzheimer's disease is early stage, the methodscan reduce or eliminate one or more symptoms characteristic of AD and/ordelay or prevent the progression from MCI to early or later stageAlzheimer's disease.

Individuals presently suffering from Alzheimer's disease can berecognized from characteristic dementia, as well as the presence of riskfactors described above. In addition, a number of diagnostic tests areavailable for identifying individuals who have AD. Individuals presentlysuffering from Alzheimer's disease can be recognized from characteristicdementia, as well as the presence of risk factors described above. Inaddition, a number of diagnostic tests are available for identifyingindividuals who have AD. These include measurement of CSF Tau,phospho-tau (pTau), sAPPα, sAPPβ, Aβ40, Aβ42 levels and/or C terminallycleaved APP fragment (APPneo). Elevated Tau, pTau, sAPPβ and/or APPneo,and/or decreased sAPPα, soluble Aβ40 and/or soluble Aβ42 levels,particularly in the context of a differential diagnosis, can signify thepresence of AD.

In certain embodiments subjects amenable to treatment may haveAlzheimer's disease. Individuals suffering from Alzheimer's disease canalso be diagnosed by Alzheimer's disease and Related DisordersAssociation (ADRDA) criteria. The NINCDS-ADRDA Alzheimer's Criteria wereproposed in 1984 by the National Institute of Neurological andCommunicative Disorders and Stroke and the Alzheimer's Disease andRelated Disorders Association (now known as the Alzheimer's Association)and are among the most used in the diagnosis of Alzheimer's disease(AD). McKhann, et al. (1984) Neurology 34(7): 939-44. According to thesecriteria, the presence of cognitive impairment and a suspected dementiasyndrome should be confirmed by neuropsychological testing for aclinical diagnosis of possible or probable AD. However, histopathologicconfirmation (microscopic examination of brain tissue) is generally usedfor a dispositive diagnosis. The NINCDS-ADRDA Alzheimer's Criteriaspecify eight cognitive domains that may be impaired in AD: memory,language, perceptual skills, attention, constructive abilities,orientation, problem solving and functional abilities). These criteriahave shown good reliability and validity.

Baseline evaluations of patient function can made using classicpsychometric measures, such as the Mini-Mental State Exam (MMSE)(Folstein et al. (1975) J. Psychiatric Research 12 (3): 189-198), andthe Alzheimer's Disease Assessment Scale (ADAS), which is acomprehensive scale for evaluating patients with Alzheimer's Diseasestatus and function (see, e.g., Rosen, et al. (1984) Am. J. Psychiatr.,141: 1356-1364). These psychometric scales provide a measure ofprogression of the Alzheimer's condition. Suitable qualitative lifescales can also be used to monitor treatment. The extent of diseaseprogression can be determined using a Mini-Mental State Exam (MMSE)(see, e.g., Folstein, et al. supra). Any score greater than or equal to25 points (out of 30) is effectively normal (intact). Below this, scorescan indicate severe (≤9 points), moderate (10-20 points) or mild (21-24points) Alzheimer's disease.

Alzheimer's disease can be broken down into various stages including: 1)Moderate cognitive decline (Mild or early-stage Alzheimer's disease), 2)Moderately severe cognitive decline (Moderate or mid-stage Alzheimer'sdisease), 3) Severe cognitive decline (Moderately severe or mid-stageAlzheimer's disease), and 4) Very severe cognitive decline (Severe orlate-stage Alzheimer's disease) as shown in Table 3.

TABLE 3 Illustrative stages of Alzheimer's disease. Moderate CognitiveDecline (Mild or early stage AD) At this stage, a careful medicalinterview detects clear-cut deficiencies in the following areas:Decreased knowledge of recent events. Impaired ability to performchallenging mental arithmetic. For example, to count backward from 100by 7s. Decreased capacity to perform complex tasks, such as marketing,planning dinner for guests, or paying bills and managing finances.Reduced memory of personal history. The affected individual may seemsubdued and withdrawn, especially in socially or mentally challengingsituations. Moderately severe cognitive decline (Moderate or mid-stageAlzheimer's disease) Major gaps in memory and deficits in cognitivefunction emerge. Some assistance with day-to-day activities becomesessential. At this stage, individuals may: Be unable during a medicalinterview to recall such important details as their current address,their telephone number, or the name of the college or high school fromwhich they graduated. Become confused about where they are or about thedate, day of the week or season. Have trouble with less challengingmental arithmetic; for example, counting backward from 40 by 4s or from20 by 2s. Need help choosing proper clothing for the season or theoccasion. Usually retain substantial knowledge about themselves and knowtheir own name and the names of their spouse or children. Usuallyrequire no assistance with eating or using the toilet. Severe cognitivedecline (Moderately severe or mid-stage Alzheimer's disease) Memorydifficulties continue to worsen, significant personality changes mayemerge, and affected individuals need extensive help with dailyactivities. At this stage, individuals may: Lose most awareness ofrecent experiences and events as well as of their surroundings.Recollect their personal history imperfectly, although they generallyrecall their own name. Occasionally forget the name of their spouse orprimary caregiver but generally can distinguish familiar from unfamiliarfaces. Need help getting dressed properly; without supervision, may makesuch errors as putting pajamas over daytime clothes or shoes on wrongfeet. Experience disruption of their normal sleep/waking cycle. Needhelp with handling details of toileting (flushing toilet, wiping anddisposing of tissue properly). Have increasing episodes of urinary orfecal incontinence. Experience significant personality changes andbehavioral symptoms, including suspiciousness and delusions (forexample, believing that their caregiver is an impostor); hallucinations(seeing or hearing things that are not really there); or compulsive,repetitive behaviors such as hand-wringing or tissue shredding. Tend towander and become lost. Very severe cognitive decline (Severe orlate-stage Alzheimer's disease) This is the final stage of the diseasewhen individuals lose the ability to respond to their environment, theability to speak, and, ultimately, the ability to control movement.Frequently individuals lose their capacity for recognizable speech,although words or phrases may occasionally be uttered. Individuals needhelp with eating and toileting and there is general incontinence.Individuals lose the ability to walk without assistance, then theability to sit without support, the ability to smile, and the ability tohold their head up. Reflexes become abnormal and muscles grow rigid.Swallowing is impaired.

In various embodiments administration of one or more agents describedherein to subjects diagnosed with Alzheimer's disease is deemedeffective when the there is a reduction in the CSF of levels of one ormore components selected from the group consisting of Tau, phospho-Tau(pTau), APPneo, soluble Aβ40, soluble Aβ42, and/or and Aβ42/Aβ40 ratio,and/or when there is a reduction of the plaque load in the brain of thesubject, and/or when there is a reduction in the rate of plaqueformation in the brain of the subject, and/or when there is animprovement in the cognitive abilities of the subject, and/or when thereis a perceived improvement in quality of life by the subject, and/orwhen there is a significant reduction in clinical dementia rating (CDR)of the subject, and/or when the rate of increase in clinical dementiarating is slowed or stopped and/or when the progression of AD is slowedor stopped (e.g., when the transition from one stage to another aslisted in Table 3 is slowed or stopped).

In certain embodiments subjects amenable to the present methodsgenerally are free of a neurological disease or disorder other thanAlzheimer's disease. For example, in certain embodiments, the subjectdoes not have and is not at risk of developing a neurological disease ordisorder such as Parkinson's disease, and/or schizophrenia, and/orpsychosis.

Active Agent(s).

The methods described herein are based, in part, on the discovery thatadministration of one or more active agents (e.g., hydantoins describedherein, or a tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said hydantoin(s), said stereoisomer(s),or said tautomer(s), or analogues, derivatives, or prodrugs thereof)find use in the treatment and/or prophylaxis of diseases characterizedby amyloid deposits in the brain, for example, mild cognitiveimpairment, Alzheimer's disease, macular degeneration, and the like.

In certain embodiments the active agent is a compound (e.g., ahydantoin) according to Formula I:

where M is

andR⁷ is selected from the group consisting of C═O, C═S, C—NH2, and C═NH,and the bond represented by the wavy line is a single bond when R⁷ isC═O, C═S, or C═NH, and a double bond when R⁷ is C—NH₂; R⁸ and R⁹ areindependently selected from the group consisting of H, alkyl,cycloalkyl, and aryl, provided that when the bond represented by thewavy line is a double bond, then R⁹ is absent; R⁰ is selected from thegroup consisting of aryl, substituted aryl, disubstituted aryl,heteroaryl, substituted heteroaryl, disubstituted heteroaryl, alkyl,haloalkyl, cycloalkyl, alkenyl, and alkynyl; X¹ is selected from thegroup consisting of C-halogen, CH, and N; A is methyl or H; R⁵ and R⁶are independently selected from halogen, H, alkyl, trichloromethyl, andtrifluoromethyl; R³ and R⁴ are independently absent or selected from thegroup consisting of alkyl, cycloalkyl, alkoxy, thioalky; and when X¹ isC, then R⁰ is not phenyl monosubstituted at the para position with—OCHF₂. Also contemplated are pharmaceutically acceptable salts thereof,tautomer thereofs, pharmaceutically acceptable salts of a tautomerthereof, an enantiomer thereof, a pharmaceutically acceptable salt of anenantiomer thereof, and the like.

In certain embodiments, the compound is a compound according to FormulaIV:

or a compound according to Formula V:

In certain embodiments, of any of the foregoing compounds, X¹ isselected from the group consisting of C-halogen, CH, and N or from thegroup consisting of CH, and N; and R⁵ and R⁶ are independently selectedhalogen. In certain embodiments, of any of the foregoing compounds, R⁷is C═NH or R⁷ is C═O.

In certain embodiments the compound is a compound according to FormulaVI:

In certain embodiments of the compound of Formula VI R⁵ and R⁶ areindependently selected halogens. In certain embodiments of the compoundof Formula VI R⁵ and R⁶ are the same halogen (e.g., both F, both Cl,etc.).

In certain embodiments the compound is a compound according to FormulaV, where said compound is a compound of Formula VII:

In certain embodiments the compound is a compound according to FormulaVIII:

In certain embodiments the compound is a compound according to FormulaIX:

In certain embodiments the compound is a compound of Formula V where R⁷is C═S.

In certain embodiments the compound is a compound where R⁷ is C—NH₂ andthe compound is a compound of Formula X:

and in certain embodiments of Formula VIII, the compound is a compoundaccording to Formula XI:

where R¹ and R² are independently absent or selected from the groupconsisting of alkyl, haloalkyl, cycloalkyl, alkenyl, alkynyl, alkoxy,thioalkyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; and X², Y, and Z are independently CH or N. In certainembodiments of any of the foregoing Formulas R⁵ and R⁶ are differenthalogens (e.g., R⁵═Cl and R⁶═F, R⁵═F and R⁶═Cl, and the like). Incertain embodiments of any of the foregoing Formulas R⁵ and R⁶ are thesame halogen (e.g., both Cl, both F, etc.).

In certain embodiments the compound is a compound of Formula XII:

In certain embodiments of any of the foregoing compounds, X¹ is CH. Incertain embodiments of any of the foregoing compounds R⁸ is H or CH₃.

In certain embodiments the compound is a compound according to FormulaXIII:

In certain embodiments the compound is a compound according to FormulaXIV:

In certain embodiments the compound is a compound according to FormulaXV:

In certain embodiments the compound is a compound according to FormulaXVI:

In certain embodiments the compound is a compound according to FormulaXVII:

In certain embodiments the compound is a compound according to FormulaXVIII:

In certain embodiments the compound is a compound according to FormulaXIX:

In certain embodiments the compound is a compound according to FormulaXX:

In certain embodiments the compound is a compound according to FormulaXXI:

In certain embodiments the compound is a compound according to FormulaXXII:

In certain embodiments the compound is a compound according to FormulaXXIII:

In certain embodiments the compound is a compound according to FormulaXXIV:

In certain embodiments the compound is a compound according to FormulaXXV:

In certain embodiments the compound is a compound according to FormulaXXVI:

In certain embodiments the compound is a compound according to FormulaXXVII:

In certain embodiments the compound is a compound according to FormulaXXVIII:

In certain embodiments the compound is a compound according to FormulaXXIX:

In certain embodiments the compound is a compound according to FormulaXXX:

In certain embodiments the compound is a compound according to FormulaXXXI:

In certain embodiments any of the foregoing Formulas expressly excludeFAH-2. In certain embodiments any of the foregoing Formulas expresslyexclude FAH-3. In certain embodiments any of the foregoing Formulasexpressly exclude FAH-2 and FAH-3.

In certain embodiments the compound is a compound according to FormulaXXXII:

or a pharmaceutically acceptable salt thereof, a tautomer thereof, apharmaceutically acceptable salt of a tautomer thereof, an enantiomerthereof, or a pharmaceutically acceptable salt of an enantiomer thereof.

In certain embodiments any of the foregoing compounds is a substantiallypure S enantiomer. In certain embodiments any of the foregoing compoundsis a substantially pure R enantiomer.

Various compounds contemplated herein include the compounds shonw inTable 4.

TABLE 4 Illustrative, but non-limiting examples of compoundscontemplated herein. FAH # Structure MW FAH-3 

381.3  FAH-1 

301.3  FAH-2 

367.3  FAH-4 

367.3  FAH-5 

329.3  FAH-6 

345.3  FAH-8 

345.3  FAH-9 

359.3  FAH-10

333.3  FAH-11

332.31 FAH-12

349.3  FAH-13

371.3  FAH-14

337.3  FAH-15

320.27 FAH-17

363.33 FAH-17 HCl

399.8  FAH-19

399.32 FAH-22

377.36 FAH-23

379.79 FAH-25

397.78 FAH-27

359.37 FAH-28

413.34With respect to these compounds pharmaceutically acceptable salts,tautomers, pharmaceutically acceptable salts of a tautomer, enantiomersthereof, and pharmaceutically acceptable salts of an enantiomer thereofare also contemplated. Additionally substantially pure S enantiomers orsubstantially pure R enantiomers of these compounds are contemplated.

Various illustrative, but non-limiting hydantoins are also shown inFIGS. 1 and 2. In certain embodiments pharmaceutically acceptable salts,tautomers, pharmaceutically acceptable salts of a tautomer, enantiomersthereof, and pharmaceutically acceptable salt of an enantiomer arecontemplated.

With respect to certain molecules in FIG. 1, without being bound to aparticular theory, it is believed that the B-ring with the methyl andthe OCHF₂ shows increased potency with a 3,4 substitution. It isbelieved that this type of a substitution pattern interacts with theTrp-76 of the BACE flap disrupting the interaction of the Tyr-71 of theflap with the Trp-76 and flipping the Tyr-71 to the left allowing it tointeract with the difluoro groups of the A-ring (see also FIG. 3).

In certain embodiments the compound is a substantially pure “S”enantiomer. In certain embodiments the compound is a substantially pure“R” enantiomer. In certain embodiments the compound binds to APP and/orto the enzyme BACE and/or to an APP/BACE complex.

Methods of preparing hydantoins such as are described herein are knownto those of skill in the art. Generally, in one approach, the relevanthydantoin (e.g., a difluoro hydantoin) would be prepared from 3,4difluoro benzaldehyde transformed to dione and condensed with urea toyield the hydantoin as described in Example 1.

Illustrative protocols for the synthesis of FAH-1, FAH-2, FAH-3, FAH-4,FAH-5, FAH-17, FAH-17 HCl salt, FAH-22, FAH-23, FAH-27, and FAH-28 (seeTable 4) are provided in Examples 1-11. Synthesis of additionalcompounds described herein are straightforward variations of thesynthesis schemes provided herein.

The various active agents and synthesis schemes are intended to beillustrative and not limiting. Using the teachings provided herein,numerous other (e.g., hydantoins or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof can be synthesized and identified byone of skill in the art.

Illustrative activity of certain APP selective BACE inhibitors describedabove is shown in Table 5.

TABLE 5 Illustrative activity of certain APP selective BACE inhibitorsdescribed herein. Primary Screen Secondary Screen BACE1 NRG1- IC50 sAPPsAPP sAPP Aβ1- BACD1 APP Compound cLogP (μM) alpha beta (α/β) 42 IC50 KdFAH-3 3.34 0.53 ↑ > 10% ↓ < 20% ↑ ≈ 10% >1 μM NA ≈0.3 μM FAH-17 3.2 0.15↑ > 20% ↓ < 50%  ↑ > 100% >1 μM >1 μM <1 μM FAH-22 0.71 ↑ > 10% ↓ < 20%↑ ≈ 50% NA NA FAH-23 0.32 ↑ > 20% ↓ < 20% ↑ > 50% NA NA FAH-27 0.13 ↑ >30% ↓ < 30%  ↑ > 100% NA NA FAH-28 0.38 ↑ > 20% ↓ < 20% ↑ > 50% NA NA

Pharmaceutical Formulations.

In certain embodiments one or more active agents described herein (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) are administered to a mammal in needthereof, e.g., to a mammal at risk for or suffering from a pathologycharacterized by abnormal processing of amyloid precursor proteins, amammal at risk for progression of MCI to Alzheimer's disease, and soforth. In certain embodiments the active agent(s) are administered toprevent or delay the onset of a pre-Alzheimer's condition and/orcognitive dysfunction, and/or to ameliorate one or more symptoms of apre-Alzheimer's cognitive dysfunction, and/or to prevent or delay theprogression of a pre-Alzheimer's condition or cognitive dysfunction toAlzheimer's disease, and/or to promote the processing of amyloidprecursor protein (APP) by a non-amyloidogenic pathway.

In certain embodiments one or more active agents described herein (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) are administered to a mammal in needthereof, e.g., to a mammal at risk for or suffering from a pathologycharacterized by abnormal processing of amyloid precursor proteins inconditions other than Alzheimer's disease of MCI. Illustrativeconditions, include, but are not limited to AD-type symptoms of patientswith Down's syndrome, glaucoma, macular degeneration (e.g., age-relatedmacular degeneration (AMD), olfactory impairment. in the treatment oftype-II diabetes, including diabetes associated with amyloidogenesis.,neurodegenerative diseases such as scrapie, bovine spongiformencaphalopathies (e.g., BSE), traumatic brain injury (“TBI”),Creutzfeld-Jakob disease and the like, type II diabetes. Otherconditions characterized by characterized by amyloidformation/deposition are contemplated. Such conditions include, but arenot limited to Huntington's Disease, medullary carcinoma of the thyroid,cardiac arrhythmias, isolated atrial amyloidosis, atherosclerosis,rheumatoid arthritis, aortic medial amyloid, prolactinomas, familialamyloid polyneuropathy, hereditary non-neuropathic systemic amyloidosis,dialysis related amyloidosis, Finnish amyloidosis, Lattice cornealdystrophy, cerebral amyloid angiopathy (e.g., Icelandic type), systemicAL amyloidosis, sporadic inclusion body myositis, cerebrovasculardementia, and the like.

The active agent(s) (e.g., hydantoins described herein) can beadministered in the “native” form or, if desired, in the form of salts,esters, amides, prodrugs, derivatives, and the like, provided the salt,ester, amide, prodrug or derivative is suitable pharmacologically, i.e.,effective in the present method(s). Salts, esters, amides, prodrugs andother derivatives of the active agents can be prepared using standardprocedures known to those skilled in the art of synthetic organicchemistry and described, for example, by March (1992) Advanced OrganicChemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y.Wiley-Interscience, and as described above.

For example, a pharmaceutically acceptable salt can be prepared for anyof the agent(s) described herein having a functionality capable offorming a salt. A pharmaceutically acceptable salt is any salt thatretains the activity of the parent compound and does not impart anydeleterious or untoward effect on the subject to which it isadministered and in the context in which it is administered.

In various embodiments pharmaceutically acceptable salts may be derivedfrom organic or inorganic bases. The salt may be a mono or polyvalention. Of particular interest are the inorganic ions, lithium, sodium,potassium, calcium, and magnesium. Organic salts may be made withamines, particularly ammonium salts such as mono-, di- and trialkylamines or ethanol amines. Salts may also be formed with caffeine,tromethamine and similar molecules.

Methods of formulating pharmaceutically active agents as salts, esters,amide, prodrugs, and the like are well known to those of skill in theart. For example, salts can be prepared from the free base usingconventional methodology that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or can be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include, but are not limited to bothorganic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvicacid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like, as well asinorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like. An acid addition saltcan be reconverted to the free base by treatment with a suitable base.Certain particularly preferred acid addition salts of the active agentsherein include halide salts, such as may be prepared using hydrochloricor hydrobromic acids. Conversely, preparation of basic salts of theactive agents of this invention are prepared in a similar manner using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or thelike. Particularly preferred basic salts include alkali metal salts,e.g., the sodium salt, and copper salts.

For the preparation of salt forms of basic drugs, the pKa of thecounterion is preferably at least about 2 pH units lower than the pKa ofthe drug. Similarly, for the preparation of salt forms of acidic drugs,the pKa of the counterion is preferably at least about 2 pH units higherthan the pKa of the drug. This permits the counterion to bring thesolution's pH to a level lower than the pH_(max) to reach the saltplateau, at which the solubility of salt prevails over the solubility offree acid or base. The generalized rule of difference in pKa units ofthe ionizable group in the active pharmaceutical ingredient (API) and inthe acid or base is meant to make the proton transfer energeticallyfavorable. When the pKa of the API and counterion are not significantlydifferent, a solid complex may form but may rapidly disproportionate(i.e., break down into the individual entities of drug and counterion)in an aqueous environment.

Preferably, the counterion is a pharmaceutically acceptable counterion.

Suitable anionic salt forms include, but are not limited to acetate,benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate,edetate, edisylate, estolate, fumarate, gluceptate, gluconate,hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate,maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,napsylate, nitrate, pamoate (embonate), phosphate and diphosphate,salicylate and disalicylate, stearate, succinate, sulfate, tartrate,tosylate, triethiodide, valerate, and the like, while suitable cationicsalt forms include, but are not limited to aluminum, benzathine,calcium, ethylene diamine, lysine, magnesium, meglumine, potassium,procaine, sodium, tromethamine, zinc, and the like.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups that are present within the molecular structureof the active agent. In certain embodiments, the esters are typicallyacyl-substituted derivatives of free alcohol groups, i.e., moieties thatare derived from carboxylic acids of the formula RCOOH where R is alky,and preferably is lower alkyl. Esters can be reconverted to the freeacids, if desired, by using conventional hydrogenolysis or hydrolysisprocedures.

Amides can also be prepared using techniques known to those skilled inthe art or described in the pertinent literature. For example, amidesmay be prepared from esters, using suitable amine reactants, or they maybe prepared from an anhydride or an acid chloride by reaction withammonia or a lower alkyl amine.

In various embodiments, the active agents identified herein (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) are useful for parenteraladministration, topical administration, oral administration, nasaladministration (or otherwise inhaled), rectal administration, or localadministration, such as by aerosol or transdermally, for prophylacticand/or therapeutic treatment of one or more of thepathologies/indications described herein (e.g., pathologiescharacterized by excess amyloid plaque formation and/or deposition orundesired amyloid or pre-amyloid processing).

In various embodiments the active agents described herein can also becombined with a pharmaceutically acceptable carrier (excipient) to forma pharmacological composition. Pharmaceutically acceptable carriers cancontain one or more physiologically acceptable compound(s) that act, forexample, to stabilize the composition or to increase or decrease theabsorption of the active agent(s). Physiologically acceptable compoundscan include, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, protection and uptake enhancerssuch as lipids, compositions that reduce the clearance or hydrolysis ofthe active agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds, particularly of use in thepreparation of tablets, capsules, gel caps, and the like include, butare not limited to binders, diluent/fillers, disintegrants, lubricants,suspending agents, and the like.

In certain embodiments, to manufacture an oral dosage form (e.g., atablet), an excipient (e.g., lactose, sucrose, starch, mannitol, etc.),an optional disintegrator (e.g. calcium carbonate,carboxymethylcellulose calcium, sodium starch glycollate, crospovidoneetc.), a binder (e.g. alpha-starch, gum arabic, microcrystallinecellulose, carboxymethylcellulose, polyvinylpyrrolidone,hydroxypropylcellulose, cyclodextrin, etc.), and an optional lubricant(e.g., talc, magnesium stearate, polyethylene glycol 6000, etc.), forinstance, are added to the active component or components (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) and the resulting composition iscompressed. Where necessary the compressed product is coated, e.g.,using known methods for masking the taste or for enteric dissolution orsustained release. Suitable coating materials include, but are notlimited to ethyl-cellulose, hydroxymethylcellulose, POLYOX® yethyleneglycol, cellulose acetate phthalate, hydroxypropylmethylcellulosephthalate, and Eudragit (Rohm & Haas, Germany; methacrylic-acryliccopolymer).

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable carrier(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the active agent(s) and on the particularphysiochemical characteristics of the active agent(s).

In certain embodiments the excipients are sterile and generally free ofundesirable matter. These compositions can be sterilized byconventional, well-known sterilization techniques. For various oraldosage form excipients such as tablets and capsules sterility is notrequired. The USP/NF standard is usually sufficient.

The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. Suitable unitdosage forms, include, but are not limited to powders, tablets, pills,capsules, lozenges, suppositories, patches, nasal sprays, injectibles,implantable sustained-release formulations, mucoadherent films, topicalvarnishes, lipid complexes, etc.

Pharmaceutical compositions comprising the active agents describedherein (e.g., hydantoins described herein, or a tautomer(s) orstereoisomer(s) thereof, or pharmaceutically acceptable salts orsolvates of said hydantoin(s), said stereoisomer(s), or saidtautomer(s), or analogues, derivatives, or prodrugs thereof) can bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. Pharmaceutical compositions can be formulated ina conventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries that facilitate processingof the active agent(s) into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

In certain embodiments, the active agents described herein areformulated for oral administration. For oral administration, suitableformulations can be readily formulated by combining the active agent(s)with pharmaceutically acceptable carriers suitable for oral deliverywell known in the art. Such carriers enable the active agent(s)described herein to be formulated as tablets, pills, dragees, caplets,lizenges, gelcaps, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients can include fillers such as sugars (e.g.,lactose, sucrose, mannitol and sorbitol), cellulose preparations (e.g.,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose), synthetic polymers (e.g., polyvinylpyrrolidone(PVP)), granulating agents; and binding agents. If desired,disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. If desired, solid dosage forms may be sugar-coated orenteric-coated using standard techniques. The preparation ofenteric-coated particles is disclosed for example in U.S. Pat. Nos.4,786,505 and 4,853,230.

For administration by inhalation, the active agent(s) are convenientlydelivered in the form of an aerosol spray from pressurized packs or anebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

In various embodiments the active agent(s) can be formulated in rectalor vaginal compositions such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides. Methods of formulating active agents for rectal or vaginaldelivery are well known to those of skill in the art (see, e.g., Allen(2007) Suppositories, Pharmaceutical Press) and typically involvecombining the active agents with a suitable base (e.g., hydrophilic(PEG), lipophilic materials such as cocoa butter or Witepsol W45,amphiphilic materials such as Suppocire AP and polyglycolized glyceride,and the like). The base is selected and compounded for a desiredmelting/delivery profile.

For topical administration the active agent(s) described herein (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) can be formulated as solutions, gels,ointments, creams, suspensions, and the like as are well-known in theart.

In certain embodiments the active agents described herein are formulatedfor systemic administration (e.g., as an injectable) in accordance withstandard methods well known to those of skill in the art. Systemicformulations include, but are not limited to, those designed foradministration by injection, e.g. subcutaneous, intravenous,intramuscular, intrathecal or intraperitoneal injection, as well asthose designed for transdermal, transmucosal oral or pulmonaryadministration. For injection, the active agents described herein can beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks solution, Ringer's solution, orphysiological saline buffer and/or in certain emulsion formulations. Thesolution(s) can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. In certain embodiments the activeagent(s) can be provided in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. For transmucosaladministration, and/or for blood/brain barrier passage, penetrantsappropriate to the barrier to be permeated can be used in theformulation. Such penetrants are generally known in the art. Injectableformulations and inhalable formulations are generally provided as asterile or substantially sterile formulation.

In addition to the formulations described previously, the activeagent(s) may also be formulated as a depot preparations. Such longacting formulations can be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the active agent(s) may be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

In certain embodiments the active agent(s) described herein can also bedelivered through the skin using conventional transdermal drug deliverysystems, i.e., transdermal “patches” wherein the active agent(s) aretypically contained within a laminated structure that serves as a drugdelivery device to be affixed to the skin. In such a structure, the drugcomposition is typically contained in a layer, or “reservoir,”underlying an upper backing layer. It will be appreciated that the term“reservoir” in this context refers to a quantity of “activeingredient(s)” that is ultimately available for delivery to the surfaceof the skin. Thus, for example, the “reservoir” may include the activeingredient(s) in an adhesive on a backing layer of the patch, or in anyof a variety of different matrix formulations known to those of skill inthe art. The patch may contain a single reservoir, or it may containmultiple reservoirs.

In one illustrative embodiment, the reservoir comprises a polymericmatrix of a pharmaceutically acceptable contact adhesive material thatserves to affix the system to the skin during drug delivery. Examples ofsuitable skin contact adhesive materials include, but are not limitedto, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir, or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the active agent(s) and any other materials that arepresent.

Alternatively, other pharmaceutical delivery systems can be employed.For example, liposomes, emulsions, and microemulsions/nanoemulsions arewell known examples of delivery vehicles that may be used to protect anddeliver pharmaceutically active compounds. Certain organic solvents suchas dimethylsulfoxide also can be employed, although usually at the costof greater toxicity.

In certain embodiments the active agent(s) described herein (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) are formulated in a nanoemulsion.Nanoemulsions include, but are not limited to oil in water (O/W)nanoemulsions, and water in oil (W/O) nanoemulsions. Nanoemulsions canbe defined as emulsions with mean droplet diameters ranging from about20 to about 1000 nm. Usually, the average droplet size is between about20 nm or 50 nm and about 500 nm. The terms sub-micron emulsion (SME) andmini-emulsion are used as synonyms.

Illustrative oil in water (O/W) nanoemulsions include, but are notlimited to: Surfactant micelles—micelles composed of small moleculessurfactants or detergents (e.g., SDS/PBS/2-propanol); Polymermicelles—micelles composed of polymer, copolymer, or block copolymersurfactants (e.g., Pluronic L64/PBS/2-propanol); Blendedmicelles—micelles in which there is more than one surfactant componentor in which one of the liquid phases (generally an alcohol or fatty acidcompound) participates in the formation of the micelle (e.g., octanoicacid/PB S/EtOH); Integral micelles—blended micelles in which the activeagent(s) serve as an auxiliary surfactant, forming an integral part ofthe micelle; and Pickering (solid phase) emulsions—emulsions in whichthe active agent(s) are associated with the exterior of a solidnanoparticle (e.g., polystyrene nanoparticles/PBS/no oil phase).

Illustrative water in oil (W/O) nanoemulsions include, but are notlimited to: Surfactant micelles—micelles composed of small moleculessurfactants or detergents (e.g., dioctyl sulfosuccinate/PBS/2-propanol,isopropylmyristate/PBS/2-propanol, etc.); Polymer micelles—micellescomposed of polymer, copolymer, or block copolymer surfactants (e.g.,PLURONIC® L121/PBS/2-propanol); Blended micelles—micelles in which thereis more than one surfactant component or in which one of the liquidphases (generally an alcohol or fatty acid compound) participates in theformation of the micelle (e.g., capric/caprylic diglyceride/PBS/EtOH);Integral micelles—blended micelles in which the active agent(s) serve asan auxiliary surfactant, forming an integral part of the micelle (e.g.,active agent/PBS/polypropylene glycol); and Pickering (solid phase)emulsions—emulsions in which the active agent(s) are associated with theexterior of a solid nanoparticle (e.g., chitosan nanoparticles/noaqueous phase/mineral oil).

As indicated above, in certain embodiments the nanoemulsions compriseone or more surfactants or detergents. In some embodiments thesurfactant is a non-anionic detergent (e.g., a polysorbate surfactant, apolyoxyethylene ether, etc.). Surfactants that find use in the presentinvention include, but are not limited to surfactants such as theTWEEN®, TRITON®, and TYLOXAPOL® families of compounds.

In certain embodiments the emulsions further comprise one or morecationic halogen containing compounds, including but not limited to,cetylpyridinium chloride. In still further embodiments, the compositionsfurther comprise one or more compounds that increase the interaction(“interaction enhancers”) of the composition with microorganisms (e.g.,chelating agents like ethylenediaminetetraacetic acid, orethylenebis(oxyethylenenitrilo)tetraacetic acid in a buffer).

In some embodiments, the nanoemulsion further comprises an emulsifyingagent to aid in the formation of the emulsion. Emulsifying agentsinclude compounds that aggregate at the oil/water interface to form akind of continuous membrane that prevents direct contact between twoadjacent droplets. Certain embodiments of the present invention featureoil-in-water emulsion compositions that may readily be diluted withwater to a desired concentration without impairing their anti-pathogenicproperties.

In addition to discrete oil droplets dispersed in an aqueous phase,certain oil-in-water emulsions can also contain other lipid structures,such as small lipid vesicles (e.g., lipid spheres that often consist ofseveral substantially concentric lipid bilayers separated from eachother by layers of aqueous phase), micelles (e.g., amphiphilic moleculesin small clusters of 50-200 molecules arranged so that the polar headgroups face outward toward the aqueous phase and the apolar tails aresequestered inward away from the aqueous phase), or lamellar phases(lipid dispersions in which each particle consists of parallelamphiphilic bilayers separated by thin films of water).

These lipid structures are formed as a result of hydrophobic forces thatdrive apolar residues (e.g., long hydrocarbon chains) away from water.The above lipid preparations can generally be described as surfactantlipid preparations (SLPs). SLPs are minimally toxic to mucous membranesand are believed to be metabolized within the small intestine (see e.g.,Hamouda et al., (1998) J. Infect. Disease 180: 1939).

In certain embodiments the emulsion comprises a discontinuous oil phasedistributed in an aqueous phase, a first component comprising an alcoholand/or glycerol, and a second component comprising a surfactant or ahalogen-containing compound. The aqueous phase can comprise any type ofaqueous phase including, but not limited to, water (e.g., dionizedwater, distilled water, tap water) and solutions (e.g., phosphatebuffered saline solution or other buffer systems). The oil phase cancomprise any type of oil including, but not limited to, plant oils(e.g., soybean oil, avocado oil, flaxseed oil, coconut oil, cottonseedoil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil,safflower oil, and sunflower oil), animal oils (e.g., fish oil), flavoroil, water insoluble vitamins, mineral oil, and motor oil. In certainembodiments, the oil phase comprises 30-90 vol % of the oil-in-wateremulsion (e.g., constitutes 30-90% of the total volume of the finalemulsion), more preferably 50-80%. The formulations need not be limitedto particular surfactants, however in certain embodiments, thesurfactant is a polysorbate surfactant (e.g., TWEEN 20®, TWEEN 40®,TWEEN 60®, and TWEEN 80®), a pheoxypolyethoxyethanol (e.g., TRITON®X-100, X-301, X-165, X-102, and X-200, and TYLOXAPOL®), or sodiumdodecyl sulfate, and the like.

In certain embodiments a halogen-containing component is present. thenature of the halogen-containing compound, in some embodiments thehalogen-containing compound comprises a chloride salt (e.g., NaCl, KCl,etc.), a cetylpyridinium halide, a cetyltrimethylammonium halide, acetyldimethylethylammonium halide, a cetyldimethylbenzylammonium halide,a cetyltributylphosphonium halide, dodecyltrimethylammonium halides,tetradecyltrimethylammonium halides, cetylpyridinium chloride,cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride,cetylpyridinium bromide, cetyltrimethylammonium bromide,cetyldimethylethylammonium bromide, cetyltributylphosphonium bromide,dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide,and the like

In certain embodiments the emulsion comprises a quaternary ammoniumcompound. Quaternary ammonium compounds include, but are not limited to,N-alkyldimethyl benzyl ammonium saccharinate,1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol; 1-Decanaminium,N-decyl-N,N-dimethyl-, chloride (or) Didecyl dimethyl ammonium chloride;2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammoniumchloride; 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzylammonium chloride; alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazoliniumchloride; alkyl bis(2-hydroxyethyl)benzyl ammonium chloride; alkyldimethyl benzyl ammonium chloride; alkyl dimethyl 3,4-dichlorobenzylammonium chloride (100% C12); alkyl dimethyl 3,4-dichlorobenzyl ammoniumchloride (50% C14, 40% C12, 10% C16); alkyl dimethyl 3,4-dichlorobenzylammonium chloride (55% C14, 23% C12, 20% C16); alkyl dimethyl benzylammonium chloride; alkyl dimethyl benzyl ammonium chloride (100% C14);alkyl dimethyl benzyl ammonium chloride (100% C16); alkyl dimethylbenzyl ammonium chloride (41% C14, 28% C12); alkyl dimethyl benzylammonium chloride (47% C12, 18% C14); alkyl dimethyl benzyl ammoniumchloride (55% C16, 20% C14); alkyl dimethyl benzyl ammonium chloride(58% C14, 28% C16); alkyl dimethyl benzyl ammonium chloride (60% C14,25% C12); alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14);alkyl dimethyl benzyl ammonium chloride (61% C12, 23% C14); alkyldimethyl benzyl ammonium chloride (65% C12, 25% C14); alkyl dimethylbenzyl ammonium chloride (67% C12, 24% C14); alkyl dimethyl benzylammonium chloride (67% C12, 25% C14); alkyl dimethyl benzyl ammoniumchloride (90% C14, 5% C12); alkyl dimethyl benzyl ammonium chloride (93%C14, 4% C12); alkyl dimethyl benzyl ammonium chloride (95% C16, 5% C18);alkyl dimethyl benzyl ammonium chloride (and) didecyl dimethyl ammoniumchloride; alkyl dimethyl benzyl ammonium chloride (as in fatty acids);alkyl dimethyl benzyl ammonium chloride (C12-C16); alkyl dimethyl benzylammonium chloride (C12-C18); alkyl dimethyl benzyl and dialkyl dimethylammonium chloride; alkyl dimethyl dimethybenzyl ammonium chloride; alkyldimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12); alkyldimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as inthe fatty acids of soybean oil); alkyl dimethyl ethylbenzyl ammoniumchloride; alkyl dimethyl ethylbenzyl ammonium chloride (60% C14); alkyldimethyl isoproylbenzyl ammonium chloride (50% C12, 30% C14, 17% C16, 3%C18); alkyl trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1%C12); alkyl trimethyl ammonium chloride (90% C18, 10% C16);alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18); Di-(C8-10)-alkyldimethyl ammonium chlorides; dialkyl dimethyl ammonium chloride; dialkyldimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkylmethyl benzyl ammonium chloride; didecyl dimethyl ammonium chloride;diisodecyl dimethyl ammonium chloride; dioctyl dimethyl ammoniumchloride; dodecyl bis(2-hydroxyethyl) octyl hydrogen ammonium chloride;dodecyl dimethyl benzyl ammonium chloride; dodecylcarbamoyl methyldimethyl benzyl ammonium chloride; heptadecyl hydroxyethylimidazoliniumchloride; hexahydro-1,3,5-thris(2-hydroxyethyl)-s-triazine;myristalkonium chloride (and) Quat RNIUM 14;N,N-Dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl dimethylbenzyl ammonium chloride; n-alkyl dimethyl ethylbenzyl ammoniumchloride; n-tetradecyl dimethyl benzyl ammonium chloride monohydrate;octyl decyl dimethyl ammonium chloride; octyl dodecyl dimethyl ammoniumchloride; octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride;oxydiethylenebis (alkyl dimethyl ammonium chloride); quaternary ammoniumcompounds, dicoco alkyldimethyl, chloride; trimethoxysily propyldimethyl octadecyl ammonium chloride; trimethoxysilyl quats, trimethyldodecylbenzyl ammonium chloride; n-dodecyl dimethyl ethylbenzyl ammoniumchloride; n-hexadecyl dimethyl benzyl ammonium chloride; n-tetradecyldimethyl benzyl ammonium chloride; n-tetradecyl dimethyl ethylbenzylammonium chloride; and n-octadecyl dimethyl benzyl ammonium chloride.

Nanoemulsion formulations and methods of making such are well known tothose of skill in the art and described for example in U.S. Pat. Nos.7,476,393, 7,468,402, 7,314,624, 6,998,426, 6,902,737, 6,689,371,6,541,018, 6,464,990, 6,461,625, 6,419,946, 6,413,527, 6,375,960,6,335,022, 6,274,150, 6,120,778, 6,039,936, 5,925,341, 5,753,241,5,698,219, an d5,152,923 and in Fanun et al. (2009) Microemulsions:Properties and Applications (Surfactant Science), CRC Press, Boca RatanFla.

In certain embodiments, one or more active agents described herein canbe provided as a “concentrate”, e.g., in a storage container (e.g., in apremeasured volume) ready for dilution, or in a soluble capsule readyfor addition to a volume of water, alcohol, hydrogen peroxide, or otherdiluent.

Extended Release (Sustained Release) Formulations.

In certain embodiments “extended release” formulations of the activeagent(s) described herein (e.g., hydantoins described herein, or atautomer(s) or stereoisomer(s) thereof, or pharmaceutically acceptablesalts or solvates of said hydantoin(s), said stereoisomer(s), or saidtautomer(s), or analogues, derivatives, or prodrugs thereof) arecontemplated. In various embodiments such extended release formulationsare designed to avoid the high peak plasma levels of intravenous andconventional immediate release oral dosage forms.

Illustrative sustained-release formulations include, for example,semipermeable matrices of solid polymers containing the therapeuticagent. Various uses of sustained-release materials have been establishedand are well known by those skilled in the art. Sustained-releasecapsules may, depending on their chemical nature, release the compoundsfor a few weeks up to over 100 days. Depending on the chemical natureand the biological stability of the therapeutic reagent, additionalstrategies for stabilization can be employed.

In certain embodiments such “extended release” formulations utilize themucosa and can independently control tablet disintegration (or erosion)and/or drug dissolution and release from the tablet over time to providea safer delivery profile. In certain embodiments the oral formulationsof active agent(s) described herein (e.g., hydantoins described herein,or a tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said hydantoin(s), said stereoisomer(s),or said tautomer(s), or analogues, derivatives, or prodrugs thereof)provide individual, repetitive doses that include a defined amount ofthe active agent that is delivered over a defined amount of time.

One illustrative sustained release formulation is a substantiallyhomogeneous composition that comprises about 0.01% to about 99% w/w, orabout 0.1% to about 95%, or about 0.1%, or about 1%, or about 2%, orabout 5%, or about 10%, or about 15%, or about 20% to about 80%, or toabout 90%, or to about 95%, or to about 97%, or to about 98%, or toabout 99%1 of the active ingredient(s) (e.g., hydantoins describedherein, or a tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said hydantoin(s), said stereoisomer(s),or said tautomer(s), or analogues, derivatives, or prodrugs thereof) andone or more mucoadhesives (also referred to herein as “bioadhesives”)that provide for adherence to the targeted mucosa of the subject(patient) and that may further comprise one or more of the following:one or more binders that provide binding of the excipients in a singletablet; one or more hydrogel forming excipients; one or more bulkingagents; one or more lubricants; one or more glidants; one or moresolubilizers; one or more surfactants; one or more flavors; one or moredisintegrants; one or more buffering excipients; one or more coatings;one or more controlled release modifiers; and one or more otherexcipients and factors that modify and control the drug's dissolution ordisintegration time and kinetics or protect the active drug fromdegradation.

In various embodiments a sustained release pharmaceutical dosage formfor oral transmucosal delivery can be solid or non-solid. In oneillustrative embodiment, the dosage form is a solid that turns into ahydrogel following contact with saliva.

Suitable excipients include, but are not limited to substances added tothe formulations that are required to produce a commercial product andcan include, but are not limited to: bulking agents, binders,surfactants, bioadhesives, lubricants, disintegrants, stabilizers,solubilizers, glidants, and additives or factors that affect dissolutionor disintegration time. Suitable excipients are not limited to thoseabove, and other suitable nontoxic pharmaceutically acceptable carriersfor use in oral formulations can be found in Remington's PharmaceuticalSciences, 17th Edition, 1985.

In certain embodiments extended release formulations of the activeagent(s) described herein for oral transmucosal drug delivery include atleast one bioadhesive (mucoadhesive) agent or a mixture of severalbioadhesives to promote adhesion to the oral mucosa during drugdelivery. In addition the bioadhesive agents may also be effective incontrolling the dosage form erosion time and/or, the drug dissolutionkinetics over time when the dosage form is wetted. Such mucoadhesivedrug delivery systems are very beneficial, since they can prolong theresidence time of the drug at the site of absorption and increase drugbioavailability. The mucoadhesive polymers forming hydrogels aretypically hydrophilic and swellable, containing numerous hydrogenbond-forming groups, like hydroxyl, carboxyl or amine, which favoradhesion. When used in a dry form, they attract water from the mucosalsurface and swell, leading to polymer/mucus interaction through hydrogenbonding, electrostatic, hydrophobic or van der Waals interaction.

Illustrative suitable mucoadhesive or bioadhesive materials, include,but are not limited to natural, synthetic or biological polymers,lipids, phospholipids, and the like. Examples of natural and/orsynthetic polymers include cellulosic derivatives (such asmethylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose,hydroxyethylmethyl cellulose, etc), natural gums (such as guar gum,xanthan gum, locust bean gum, karaya gum, veegum etc.), polyacrylates(such as CARBOPOL®, polycarbophil, etc), alginates, thiol-containingpolymers, POLYOX® yethylenes, polyethylene glycols (PEG) of allmolecular weights (preferably between 1000 and 40,000 Da, of anychemistry, linear or branched), dextrans of all molecular weights(preferably between 1000 and 40,000 Da of any source), block copolymers,such as those prepared by combinations of lactic and glycolic acid (PLA,PGA, PLGA of various viscosities, molecular weights andlactic-to-glycolic acid ratios) polyethylene glycol-polypropylene glycolblock copolymers of any number and combination of repeating units (suchas PLURONICS®, TEKTRONIX® or GENAPOL® block copolymers), combination ofthe above copolymers either physically or chemically linked units (forexample PEG-PLA or PEG-PLGA copolymers) mixtures. Preferably thebioadhesive excipient is selected from the group of polyethyleneglycols, POLYOX® yethylenes, polyacrylic acid polymers, such asCARBOPOL® (such as CARBOPOL® 71G, 934P, 971P, 974P, and the like) andpolycarbophils (such as NOVEON® AA-1, NOVEON® CA-1, NOVEON® CA-2, andthe like), cellulose and its derivatives and most preferably it ispolyethylene glycol, carbopol, and/or a cellulosic derivative or acombination thereof.

In certain embodiments the mucoadhesive/bioadhesive excipient istypically present at 1-50% w/w, preferably 1-40% w/w or most preferablybetween 5-30% w/w. A particular formulation may contain one or moredifferent bioadhesives in any combination.

In certain embodiments the formulations for oral transmucosal drugdelivery also include a binder or mixture of two or more binders whichfacilitate binding of the excipients into a single dosage form.Illustrative binders include, binders selected from the group consistingof cellulosic derivatives (such as methylcellulose, carboxymethylcellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, etc.),polyacrylates (such as CARBOPOL®, polycarbophil, etc.), POVIDONE® (allgrades), POLYOX®® of any molecular weight or grade, irradiated or not,starch, polyvinylpyrrolidone (PVP), AVICEL®, and the like. In certainembodiments the binder is typically present at 0.5-60% w/w, preferably1-30% w/w and most preferably 1.5-15% w/w.

In certain embodiments the formulations also include at least onehydrogel-forming excipient. Illustrative hydrogel forming excipientsinclude, but are not limited to those selected from the group consistingof polyethylene glycols and other polymers having an ethylene glycolbackbone, whether homopolymers or cross linked heteropolymers, blockcopolymers using ethylene glycol units, such as POLYOX® yethylenehomopolymers (such as POLYOX®® N10/MW=100,000 POLYOX®-80/MW=200,000;POLYOX® 1105/MW=900,000; POLYOX®-301/MW=4,000,000;POLYOX®-303/MW=7,000,000, POLYOX® WSR-N-60K, all of which are tradenamesof Union Carbide), hydroxypropylmethylcellylose (HPMC) of all molecularweights and grades (such as METOLOSE® 90SH50000, METOLOSE® 90SH30000,all of which are tradenames of Shin-Etsu Chemical company), Poloxamers(such as LUTROL® F-68, LUTROL® F-127, F-105 etc., all tradenames of BASFChemicals), GENAPOL®, polyethylene glycols (PEG, such as PEG-1500,PEG-3500, PEG-4000, PEG-6000, PEG-8000, PEG-12000, PEG-20,000, etc.),natural gums (xanthan gum, locust bean gum, etc.) and cellulosederivatives (HC, HMC, HMPC, HPC, CP, CMC), polyacrylic acid-basedpolymers either as free or cross-linked and combinations thereof,biodegradable polymers such as poly lactic acids, polyglycolic acids andany combination thereof, whether a physical blend or cross-linked. Incertain embodiments, the hydrogel components may be cross-linked. Thehydrogel forming excipient(s) are typically present at 0.1-70% w/w,preferably 1-50% w/w or most preferably 1-30% w/w.

In certain embodiments the formulations may also include at least onecontrolled release modifier which is a substance that upon hydration ofthe dosage form will preferentially adhere to the drug molecules andthus reduce the rate of its diffusion from the oral dosage form. Suchexcipients may also reduce the rate of water uptake by the formulationand thus enable a more prolonged drug dissolution and release from thetablet. In general the selected excipient(s) are lipophilic and capableof naturally complexing to the hydrophobic or lipophilic drugs. Thedegree of association of the release modifier and the drug can be variedby altering the modifier-to-drug ratio in the formulation. In addition,such interaction may be appropriately enhanced by the appropriatecombination of the release modifier with the active drug in themanufacturing process. Alternatively, the controlled release modifiermay be a charged polymer either synthetic or biopolymer bearing a netcharge, either positive or negative, and which is capable of binding tothe active via electrostatic interactions thus modifying both itsdiffusion through the tablet and/or the kinetics of its permeationthrough the mucosal surface. Similarly to the other compounds mentionedabove, such interaction is reversible and does not involve permanentchemical bonds with the active. In certain embodiments the controlledrelease modifier may typically be present at 0-80% w/w, preferably 1-20%w/w, most preferably 1-10% w/w.

In various embodiments the extended release formulations may alsoinclude other conventional components required for the development oforal dosage forms, which are known to those skilled in the art. Thesecomponents may include one or more bulking agents (such as lactose USP,Starch 1500, mannitol, sorbitol, malitol or other non-reducing sugars;microcrystalline cellulose (e.g., AVICEL®), dibasic calcium phosphatedehydrate, sucrose, and mixtures thereof), at least one solubilizingagent(s) (such as cyclodextrins, pH adjusters, salts and buffers,surfactants, fatty acids, phospholipids, metals of fatty acids etc.),metal salts and buffers organic (such as acetate, citrate, tartrate,etc.) or inorganic (phosphate, carbonate, bicarbonate, borate, sulfate,sulfite, bisulfite, metabisulfite, chloride, etc.), salts of metals suchas sodium, potassium, calcium, magnesium, etc.), at least one lubricant(such as stearic acid and divalent cations of, such as magnesiumstearate, calcium stearate, etc., talc, glycerol monostearate and thelike), one or more glidants (such as colloidal silicon dioxide,precipitated silicon dioxide, fumed silica (CAB-O-SIL® M-5P, trademarkof Cabot Corporation), stearowet and sterotex, silicas (such as SILOID®and SILOX® silicas—trademarks of Grace Davison Products,Aerosil—trademark of Degussa Pharma), higher fatty acids, the metalsalts thereof, hydrogenated vegetable oils and the like), flavors orsweeteners and colorants (such as aspartame, mannitol, lactose, sucrose,other artificial sweeteners; ferric oxides and FD&C lakes), additives tohelp stabilize the drug substance from chemical of physical degradation(such as anti-oxidants, anti-hydrolytic agents, aggregation-blockersetc. Anti-oxidants may include BHT, BHA, vitamins, citric acid, EDTA,sodium bisulfate, sodium metabisulfate, thiourea, methionine,surfactants, amino-acids, such as arginine, glycine, histidine,methionine salts, pH adjusters, chelating agents and buffers in the dryor solution form), one or more excipients that may affect tabletdisintegration kinetics and drug release from the tablet, and thuspharmacokinetics (disintegrants such as those known to those skilled inthe art and may be selected from a group consisting of starch,carboxy-methycellulose type or crosslinked polyvinyl pyrrolidone (suchas cross-povidone, PVP-XL), alginates, cellulose-based disintegrants(such as purified cellulose, methylcellulose, crosslinked sodium carboxymethylcellulose (Ac-Di-Sol) and carboxy methyl cellulose), lowsubstituted hydroxypropyl ethers of cellulose, microcrystallinecellulose (such as AVICEL®), ion exchange resins (such as AMBRELITE® IPR88), gums (such as agar, locust bean, karaya, pectin and tragacanth),guar gums, gum karaya, chitin and chitosan, smecta, gellan gum,isapghula husk, polacrillin potassium (Tulsion³³⁹), gas-evolvingdisintegrants (such as citric acid and tartaric acid along with thesodium bicarbonate, sodium carbonate, potassium bicarbonate or calciumcarbonate), sodium starch glycolate (such as EXPLOTAB® and PRIMOGEL®),starch DC and the likes, at least one biodegradable polymer of any typeuseful for extended drug release. Exemplary polymer compositionsinclude, but are not limited to, polyanhydrides and co-polymers oflactic acid and glycolic acid, poly(dl-lactide-co-glycolide) (PLGA),poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polyorthoesters,proteins, and polysaccharides.

In certain embodiments, the active agent(s) can be chemically modifiedto significantly modify the pharmacokinetics in plasma. This may beaccomplished for example by conjugation with poly(ethylene glycol)(PEG), including site-specific PEGylation. PEGylation, which may improvedrug performance by optimizing pharmacokinetics, decreasingimmunogenicity and dosing frequency.

Methods of making a formulation of the active agent(s) described herein(e.g., hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) for GI or oral transmucosal deliveryare also provided. One method includes the steps of powder grinding, drypowder mixing and tableting via direct compression. Alternatively, a wetgranulation process may be used. Such a method (such as high sheargranulation process) involves mixing the active ingredient and possiblysome excipients in a mixer. The binder may be one of the excipientsadded in the dry mix state or dissolved in the fluid used forgranulating. The granulating solution or suspension is added to the drypowders in the mixer and mixed until the desired characteristics areachieved. This usually produces a granule that will be of suitablecharacteristics for producing dosage forms with adequate dissolutiontime, content uniformity, and other physical characteristics. After thewet granulation step, the product is most often dried and/or then milledafter drying to get a major percentage of the product within a desiredsize range. Sometimes, the product is dried after being wet sized usinga device such as an oscillating granulator, or a mill. The drygranulation may then processed to get an acceptable size range by firstscreening with a sieving device, and then milling the oversizedparticles.

Additionally, the formulation may be manufactured by alternativegranulation processes, all known to those skilled in the art, such asspray fluid bed granulation, extrusion and spheronization or fluid bedrotor granulation.

Additionally, the tablet dosage form of the active agent(s) describedherein may be prepared by coating the primary tablet manufactured asdescribed above with suitable coatings known in the art. Such coatingsare meant to protect the active cores against damage (abrasion,breakage, dust formation) against influences to which the cores areexposed during transport and storage (atmospheric humidity, temperaturefluctuations), and naturally these film coatings can also be colored.The sealing effect of film coats against water vapor is expressed by thewater vapor permeability. Coating may be performed by one of theavailable processes such as Wurster coating, dry coating, film coating,fluid bed coating, pan coating, etc. Typical coating materials includepolyvinyl pyrrolidone (PVP), polyvinyl pyrrolidone vinyl acetatecopolymer (PVPVA), polyvinyl alcohol (PVA), polyvinylalcohol/polyethylene glycol copolymer (PVA/PEG), cellulose acetatephthalate, ethyl cellulose, gellan gum, maltodextrin, methacrylates,methyl cellulose, hydroxyl propyl methyl cellulose (HPMC of all gradesand molecular weights), carrageenan, shellac and the like.

In certain embodiments the tablet core comprising the active agent(s)described herein can be coated with a bioadhesive and/or pH resistantmaterial to enable material, such as those defined above, to improvebioadhesion of the tablet in the sublingual cavity.

In certain embodiments, the active agent(s) described herein (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) are formulated as inclusion complexes.While not limited to cyclodextrin inclusion complexes, it is noted thatcyclodextrin is the agent most frequently used to form pharmaceuticalinclusion complexes. Cyclodextrins (CD) are cyclic oligomers of glucose,that typically contain 6, 7, or 8 glucose monomers joined by a-1,4linkages. These oligomers are commonly called α-CD, β-CD, and γ-CD,respectively. Higher oligomers containing up to 12 glucose monomers areknown, and contemplated to in the formulations described herein.Functionalized cyclodextrin inclusion complexes are also contemplated.Illustrative, but non-limiting functionalized cyclodextrins include, butare not limited to sulfonates, sulfonates and sulfinates, ordisulfonates of hydroxybutenyl cyclodextrin; sulfonates, sulfonates andsulfinates, or disulfonates of mixed ethers of cyclodextrins where atleast one of the ether substituents is hydroxybutenyl cyclodextrin.Illustrative cyclodextrins include a polysaccharide ether whichcomprises at least one 2-hydroxybutenyl substituent, wherein the atleast one hydroxybutenyl substituent is sulfonated and sulfinated, ordisulfonated, and an alkylpolyglycoside ether which comprises at leastone 2-hydroxybutenyl substituent, wherein the at least onehydroxybutenyl substituent is sulfonated and sulfinated, ordisulfonated. In various embodiments inclusion complexes formed betweensulfonated hydroxybutenyl cyclodextrins and one or more of the activeagent(s) described herein are contemplated. Methods of preparingcyclodextrins, and cyclodextrin inclusion complexes are found forexample in U.S. Patent Publication No: 2004/0054164 and the referencescited therein and in U.S. Patent Publication No: 2011/0218173 and thereferences cited therein.

Pharmacokinetics (PK) and Formulation Attributes

One advantage of the extended (controlled) release oral (GI ortransmucosal) formulations described herein is that they can maintainthe plasma drug concentration within a targeted therapeutic window for alonger duration than with immediate-release formulations, whether soliddosage forms or liquid-based dosage forms. The high peak plasma levelstypically observed for such conventional immediate release formulationswill be blunted by the prolonged release of the drug over 1 to 12 hoursor longer. In addition, a rapid decline in plasma levels will be avoidedsince the drug will continually be crossing from the oral cavity intothe bloodstream during the length of time of dissolution of the tablet,thus providing plasma pharmacokinetics with a more stable plateau. Inaddition, the dosage forms described herein may improve treatment safetyby minimizing the potentially deleterious side effects due to thereduction of the peaks and troughs in the plasma drug pharmacokinetics,which compromise treatment safety.

In various embodiments the oral transmucosal formulations of the activeagent(s) described herein designed to avoid the high peak plasma levelsof intravenous and conventional immediate release oral dosage forms byutilizing the mucosa and by independently controlling both tabletdisintegration (or erosion) and drug dissolution and release from thetablet over time to provide a safer delivery profile. The oralformulations described herein provide individual, repetitive doses thatinclude a defined amount of the active agent.

An advantage of the bioadhesive oral transmucosal formulations describedherein is that they exhibit highly consistent bioavailability and canmaintain the plasma drug concentration within a targeted therapeuticwindow with significantly lower variability for a longer duration thancurrently available dosage forms, whether solid dosage forms or IVdosage forms. In addition, a rapid decline in plasma levels is avoidedsince the drug is continually crossing from the oral cavity or GI tractinto the bloodstream during the length of time of dissolution of thetablet or longer, thus providing plasma pharmacokinetics with anextended plateau phase as compared to the conventional immediate releaseoral dosage forms. Further, the dosage forms described herein canimprove treatment safety by minimizing the potentially deleterious sideeffects due to the relative reduction of the peaks and troughs in theplasma drug pharmacokinetics, which compromise treatment safety and istypical of currently available dosage forms.

In various embodiments bioadhesive formulations described herein can bedesigned to manipulate and control the pharmacokinetic profile of theactive agent(s) described herein. As such, the formulations can beadjusted to achieve ‘slow’ disintegration times (and erosion kineticprofiles) and slow drug release and thus enable very prolongedpharmacokinetic profiles that provide sustained drug action. Althoughsuch formulations may be designed to still provide a fast onset, theyare mostly intended to enable the sustained drug PK and effect whilemaintaining the other performance attributes of the tablet such asbioadhesion, reproducibility of action, blunted C_(max), etc.

The performance and attributes of the bioadhesive transmucosalformulations of this invention are independent of the manufacturingprocess. A number of conventional, well-established and known in the artprocesses can be used to manufacture the formulations of the presentinvention (such as wet and dry granulation, direct compression, etc.)without impacting the dosage form physicochemical properties or in vivoperformance.

An illustrative mathematical ratio that demonstrates the prolongedplateau phase of the measured blood plasma levels of the active agent(s)described herein, following administration of the dosage forms of theinvention is the term “Optimal Therapeutic Targeting Ratio” or “OTTR”,which represents the average time that the drug is present attherapeutic levels, defined as time within which the drug plasmaconcentration is maintained above 50% of C_(max) normalized by thedrug's elimination half-life multiplied by the ratio of the C_(max)obtained in the dosage form of interest over the normalized C_(max)following IV administration of equivalent doses. In certain embodimentsthe OTTR can be calculated by the formula:

OTTR=(C ^(IV) _(max) /C _(max))×(Dose/Dose^(IV))(Time above 50% of C_(max))/(Terminal^(IV) elimination half-life of the drug).

In certain embodiments the OTTR is greater than about 15, or greaterthan about 20, or greater than about 25, or greater than about 30, orgreater than about 40, or greater than about 50.

Administration

In certain embodiments one or more active agents described herein (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) are administered to a mammal in needthereof, e.g., to a mammal at risk for or suffering from a pathologycharacterized by abnormal processing of amyloid precursor proteins, amammal at risk for progression of MCI to Alzheimer's disease, and soforth. In certain embodiments the active agent(s) are administered toprevent or delay the onset of a pre-Alzheimer's cognitive dysfunction,and/or to ameliorate one or more symptoms of a pre-Alzheimer's cognitivedysfunction, and/or to prevent or delay the progression of apre-Alzheimer's condition or cognitive dysfunction to Alzheimer'sdisease, and/or to promote the processing of amyloid precursor protein(APP) by a non-amyloidogenic pathway. In certain embodiments one or moreactive agent(s) are administered for the treatment of early stage, midstage, or late-stage Alzheimer's disease, e.g., to reduce the severityof the disease, and/or to ameliorate one or more symptoms of thedisease, and/or to slow the progression of the disease.

In various embodiments the active agent(s) described herein (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) can be administered by any of a numberof routes. Thus, for example they can be administered orally,parenterally, (intravenously (IV), intramuscularly (IM), depo-IM,subcutaneously (SQ), and depo-SQ), sublingually, intranasally(inhalation), intrathecally, transdermally (e.g., via transdermalpatch), topically, ionophoretically or rectally. Typically the dosageform is selected to facilitate delivery to the brain (e.g., passagethrough the blood brain barrier). In this context it is noted that thecompounds described herein are readily delivered to the brain. Dosageforms known to those of skill in the art are suitable for delivery ofthe compound.

In various embodiments the active agent(s) are administered in anamount/dosage regimen sufficient to exert a prophylactically and/ortherapeutically useful effect in the absence of undesirable side effectson the subject treated (or with the presence of acceptable levels and/ortypes of side effects). The specific amount/dosage regimen will varydepending on the weight, gender, age and health of the individual; theformulation, the biochemical nature, bioactivity, bioavailability andthe side effects of the particular compound.

In certain embodiments the therapeutically or prophylactically effectiveamount may be determined empirically by testing the agent(s) in known invitro and in vivo model systems for the treated disorder. Atherapeutically or prophylactically effective dose can be determined byfirst administering a low dose, and then incrementally increasing untila dose is reached that achieves the desired effect with minimal or noundesired side effects.

In certain embodiments, when administered orally, an administered amountof the agent(s) described herein effective to prevent or delay the onsetof a pre-Alzheimer's cognitive dysfunction, and/or to ameliorate one ormore symptoms of a pre-Alzheimer's cognitive dysfunction, and/or toprevent or delay the progression of a pre-Alzheimer's condition orcognitive dysfunction to Alzheimer's disease, and/or to promote theprocessing of amyloid precursor protein (APP) by a non-amyloidogenicpathway, and/or to treat or prevent AD ranges from about 0.1 mg/day toabout 500 mg/day or about 1,000 mg/day, or from about 0.1 mg/day toabout 200 mg/day, for example, from about 1 mg/day to about 100 mg/day,for example, from about 5 mg/day to about 50 mg/day. In someembodiments, the subject is administered the compound at a dose of about0.05 to about 0.50 mg/kg, for example, about 0.05 mg/kg, 0.10 mg/kg,0.20 mg/kg, 0.33 mg/kg, 0.50 mg/kg. It is understood that while apatient may be started at one dose, that dose may be varied (increasedor decreased, as appropriate) over time as the patient's conditionchanges. Depending on outcome evaluations, higher doses may be used. Forexample, in certain embodiments, up to as much as 1000 mg/day can beadministered, e.g., 5 mg/day, 10 mg/day, 25 mg/day, 50 mg/day, 100mg/day, 200 mg/day, 300 mg/day, 400 mg/day, 500 mg/day, 600 mg/day, 700mg/day, 800 mg/day, 900 mg/day or 1000 mg/day.

In various embodiments, active agent(s) described herein can beadministered parenterally, for example, by IV, IM, depo-IM, SC, ordepo-SC. In certain embodiments when administered parenterally, atherapeutically effective amount of about 0.5 to about 100 mg/day,preferably from about 5 to about 50 mg daily can be delivered. When adepot formulation is used for injection once a month or once every twoweeks, the dose in certain embodiments can be about 0.5 mg/day to about50 mg/day, or a monthly dose of from about 15 mg to about 1,500 mg. Inpart because of the forgetfulness of the patients with Alzheimer'sdisease, it is preferred that the parenteral dosage form be a depoformulation.

In various embodiments, the active agent(s) described herein can beadministered sublingually. In some embodiments, when given sublingually,the compounds and/or analogs thereof can be given one to four timesdaily in the amounts described above for IM administration.

In various embodiments, the active agent(s) described herein can beadministered intranasally. When given by this route, the appropriatedosage forms are a nasal spray or dry powder, as is known to thoseskilled in the art. In certain embodiments, the dosage of compoundand/or analog thereof for intranasal administration is the amountdescribed above for IM administration.

In various embodiments, the active agent(s) described herein can beadministered intrathecally. When given by this route the appropriatedosage form can be a parenteral dosage form as is known to those skilledin the art. In certain embodiments, the dosage of compound and/or analogthereof for intrathecal administration is the amount described above forIM administration.

In certain embodiments, the active agent(s) described herein can beadministered topically. When given by this route, the appropriate dosageform is a cream, ointment, or patch. When administered topically, thedosage is from about 1.0 mg/day to about 200 mg/day. Because the amountthat can be delivered by a patch is limited, two or more patches may beused. The number and size of the patch is not important as long as atherapeutically effective amount of compound be delivered as is known tothose skilled in the art. The compound can be administered rectally bysuppository as is known to those skilled in the art. In certainembodiments, when administered by suppository, the therapeuticallyeffective amount is from about 1.0 mg to about 500 mg.

In various embodiments, the active agent(s) described herein can beadministered by implants as is known to those skilled in the art. Whenadministering the compound by implant, the therapeutically effectiveamount is the amount described above for depot administration.

In various embodiments, the active agent(s) described herein thereof canbe enclosed in multiple or single dose containers. The enclosed agent(s)can be provided in kits, for example, including component parts that canbe assembled for use. For example, an active agent in lyophilized formand a suitable diluent may be provided as separated components forcombination prior to use. A kit may include an active agent and a secondtherapeutic agent for co-administration. The active agent and secondtherapeutic agent may be provided as separate component parts. A kit mayinclude a plurality of containers, each container holding one or moreunit dose of the compounds. The containers are preferably adapted forthe desired mode of administration, including, but not limited totablets, gel capsules, sustained-release capsules, and the like for oraladministration; depot products, pre-filled syringes, ampules, vials, andthe like for parenteral administration; and patches, medipads, creams,and the like for topical administration, e.g., as described herein.

In various embodiments the dosage forms can be administered to thesubject 1, 2, 3, or 4 times daily. In certain embodiments it ispreferred that the compound be administered either three or fewer times,more preferably once or twice daily. In certain embodiments, it ispreferred that the agent(s) be administered in oral dosage form.

It should be apparent to one skilled in the art that the exact dosageand frequency of administration will depend on the particular conditionbeing treated, the severity of the condition being treated, the age,weight, general physical condition of the particular patient, and othermedication the individual may be taking as is well known toadministering physicians who are skilled in this art.

While the compositions and methods are described herein with respect touse in humans, they are also suitable for animal, e.g., veterinary use.Thus certain organisms (subjects) contemplated herein include, but arenot limited to humans, non-human primates, canines, equines, felines,porcines, ungulates, largomorphs, and the like.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

Combination Therapies

In certain embodiments, the active agent(s) described herein (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) can be used in combination with othertherapeutic agents or approaches used to treat or prevent diseasescharacterized by amyloid deposits in the brain, including MCI and/or AD.Accordingly, in certain embodiments, a pharmaceutical compositioncomprising at least active agent described herein (e.g., a hydantoindescribed herein, or a tautomer or stereoisomer thereof, orpharmaceutically acceptable salts or solvate of said hydantoin, saidstereoisomer, or said tautomer, or an analogue, derivative, or prodrugthereof) one together with at least one additional therapeutic agent,and a pharmaceutically acceptable carrier or diluent is contemplated. Incertain embodiments a therapeutic or prophylactic method comprisingadministering at least active agent described herein in conjunction withat least one additional therapeutic agent is contemplated.

In certain embodiments non-limiting examples of additional therapeuticagents include, but are not limited to disulfiram and/or analoguesthereof, honokiol and/or analogues thereof, tropisetron and/or analoguesthereof, nimetazepam and/or analogues thereof (see, e.g., U.S. Ser. No.13/213,960 (U.S. Patent Publication No: US-2012-0071468-A1), andPCT/US2011/048472 (PCT Publication No: WO 2012/024616) which areincorporated herein by reference for the compounds described therein),tropinol-esters and/or related esters and/or analogues thereof (see,e.g., U.S. Ser. No. 61/514,381, which is incorporated herein byreference for the compounds described herein), TrkA kinase inhibitors(e.g., ADDN-1351) and/or analogues thereof (see, e.g., U.S. Ser. No.61/525,076, which is incorporated herein by reference for the compoundsdescribed therein), D2 receptor agonists and alpha1-adrenergic receptorantagonists, and APP-specific BACE Inhibitors (ASBIs) as describedand/or claimed in U.S. Ser. No. 61/728,688, filed on Nov. 20, 2012 whichis incorporated herein by reference for the active agents describedherein including, but not limited to galangin, a galangin prodrug,rutin, a rutin prodrug, and other flavonoids and flavonoid prodrugs asdescribed or claimed therein.

Non-limiting examples of additional therapeutic agents include drugsselected from the group consisting of: (a) drugs useful for thetreatment of Alzheimer's disease and/or drugs useful for treating one ormore symptoms of Alzheimer's disease, (b) drugs useful for inhibitingthe synthesis Aβ, and (c) drugs useful for treating neurodegenerativediseases. Additional non-limiting examples of additional therapeuticagents for use in combination with the compounds (e.g., hydantoins)described herein include drugs useful for the treatment, prevention,delay of onset, amelioration of any pathology associated with Aβ and/ora symptom thereof. Non-limiting examples of pathologies associated withAβ include: Alzheimer's disease, Down's syndrome, Parkinson's disease,memory loss, memory loss associated with Alzheimer's disease, memoryloss associated with Parkinson's disease, attention deficit symptoms,attention deficit symptoms associated with Alzheimer's disease,Parkinson's disease, and/or Down's syndrome, dementia, stroke,microgliosis and brain inflammation, pre-senile dementia, seniledementia, dementia associated with Alzheimer's disease, Parkinson'sdisease, and/or Down's syndrome, progressive supranuclear palsy,cortical basal degeneration, neurodegeneration, olfactory impairment,olfactory impairment associated with Alzheimer's disease, Parkinson'sdisease, and/or Down's syndrome, β-amyloid angiopathy, cerebral amyloidangiopathy, hereditary cerebral hemorrhage, mild cognitive impairment(“MCI”), glaucoma, amyloidosis, type II diabetes, hemodialysiscomplications (from β.sub.2 microglobulins and complications arisingtherefrom in hemodialysis patients), scrapie, bovine spongiformencephalitis, traumatic brain injury (“TBI”), and Creutzfeld-Jakobdisease, comprising administering to said patient at least one hydantoincompound described herein, or a tautomer or isomer thereof; orpharmaceutically acceptable salt or solvate of said compound or saidtautomer, in an amount effective to inhibit said pathology orpathologies.

In certain embodiments such additional therapeutic agents include, butare not limited to acetylcholinesterase inhibitors (including withoutlimitation, e.g., (−)-phenserine enantiomer, tacrine, ipidacrine,galantamine, donepezil, icopezil, zanapezil, rivastigmine, huperzine A,phenserine, physostigmine, neostigmine, pyridostigmine, ambenonium,demarcarium, edrophonium, ladostigil and ungeremine); NMDA receptorantagonist (including without limitations e.g., Memantine); muscarinicreceptor agonists (including without limitation, e.g., Talsaclidine,AF-102B, AF-267B (NGX-267)); nicotinic receptor agonists (includingwithout limitation, e.g., Ispronicline (AZD-3480)); beta-secretaseinhibitors (including without limitations e.g., thiazolidinediones,including rosiglitazone and pioglitazone); gamma-secretase inhibitors(including without limitation, e.g., semagacestat (LY-450139), MK-0752,E-2012, BMS-708163, PF-3084014, begacestat (GSI-953), and NIC5-15);inhibitors of Aβ aggregation (including without limitation, e.g.,Clioquinol (PBT1), PBT2, tramiprosate (homotaurine), Scyllo-inositol(a.k.a., scyllo-cyclohexanehexol, AZD-103 and ELND-005), passiveimmunotherapy with Aβ fragments (including without limitations e.g.,Bapineuzemab) and Epigallocatechin-3-gallate (EGCg)); anti-inflammatoryagents such as cyclooxygenase II inhibitors; anti-oxidants such asVitamin E and ginkolides; immunological approaches, such as, forexample, immunization with Aβ peptide or administration of anti-Aβpeptide antibodies; statins; and direct or indirect neurotrophic agentssuch as Cerebrolysin™, AIT-082 (Emilieu, 2000, Arch. Neurol. 57:454),Netrin (Luorenco (2009) Cell Death Differ., 16: 655-663), Netrinmimetics, NGF, NGF mimetics, BDNF and other neurotrophic agents of thefuture, agents that promote neurogenesis e.g. stem cell therapy. Furtherpharmacologic agents useful in the treatment or prevention diseasescharacterized by amyloid deposits in the brain, including MCI and/or AD,are described, e.g., in Mangialasche, et al. (2010) Lancet Neurol.,9:702-716.

In certain embodiments, additional non-limiting examples of additionaltherapeutic agents for use in combination with compounds describedherein include: muscarinic antagonists (e.g., m₁ agonists (such asacetylcholine, oxotremorine, carbachol, or McNa343), or m₂ antagonistscholinesterase inhibitors (e.g., acetyl- and/or butyrylchlolinesteraseinhibitors such as donepezil (Aricept®), galantamine (Razadyne®), andrivastigimine (Exelon®); N-methyl-D-aspartate receptor antagonists(e.g., NAMENDA® (memantine HCl); combinations of cholinesteraseinhibitors and N-methyl-D-aspartate receptor antagonists; gammasecretase modulators; gamma secretase inhibitors; non-steroidalanti-inflammatory agents; anti-inflammatory agents that can reduceneuroinflammation; anti-amyloid antibodies (such as bapineuzemab,Wyeth/Elan); vitamin E; nicotinic acetylcholine receptor agonists; CB 1receptor inverse agonists or CB 1 receptor antagonists; antibiotics;growth hormone secretagogues; histamine H3 antagonists; AMPA agonists;PDE4 inhibitors; GABA_(A) inverse agonists; inhibitors of amyloidaggregation; glycogen synthase kinase beta inhibitors; promoters ofalpha secretase activity; PDE-10 inhibitors; Tau kinase inhibitors(e.g., GSK3beta inhibitors, cdk5 inhibitors, or ERK inhibitors); Tauaggregation inhibitors (e.g., REMBER®; RAGE inhibitors (e.g., TTP 488(PF-4494700)); anti-Aβ vaccine; APP ligands; agents that upregulateinsulin, cholesterol lowering agents such as HMG-CoA reductaseinhibitors (for example, statins such as Atorvastatin, Fluvastatin,Lovastatin, Mevastatin, Pitavastatin, Pravastatin, Rosuvastatin,Simvastatin) and/or cholesterol absorption inhibitors (such asEzetimibe), or combinations of HMG-CoA reductase inhibitors andcholesterol absorption inhibitors (such as, for example, VYTORIN®);fibrates (such as, for example, clofibrate, Clofibride, Etofibrate, andAluminium Clofibrate); combinations of fibrates and cholesterol loweringagents and/or cholesterol absorption inhibitors; nicotinic receptoragonists; niacin; combinations of niacin and cholesterol absorptioninhibitors and/or cholesterol lowering agents (e.g., SIMCOR®(niacin/simvastatin, available from Abbott Laboratories, Inc.); LXRagonists; LRP mimics; H3 receptor antagonists; histone deacetylaseinhibitors; hsp90 inhibitors; 5-HT4 agonists (e.g., PRX-03140 (EpixPharmaceuticals)); 5-HT6 receptor antagonists; mGluR1 receptormodulators or antagonists; mGluR5 receptor modulators or antagonists;mGluR2/3 antagonists; Prostaglandin EP2 receptor antagonists; PAI-1inhibitors; agents that can induce Abeta efflux such as gelsolin;Metal-protein attenuating compound (e.g., PBT2); and GPR3 modulators;and antihistamines such as Dimebolin (e.g., DIMEBON®, Pfizer).

Accordingly certain embodiments provide a pharmaceutical compositioncomprising an effective amount of one or more hydantoins describedherein and an additional therapeutic agent, and/or a method of treatmentor prophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method isdisulfiram and/or analogues thereof (see, e.g., U.S. Ser. No. 13/213,960(U.S. Patent Publication No: US-2012-0071468-A1), and PCT/US2011/048472(PCT Publication No: WO 2012/024616)).

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method is honokioland/or analogues thereof (see, e.g., U.S. Ser. No. 13/213,960 (U.S.Patent Publication No: US-2012-0071468-A1), and PCT/US2011/048472 (PCTPublication No: WO 2012/024616)).

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method istropisetron and/or analogues thereof (see, e.g., U.S. Ser. No.13/213,960 (U.S. Patent Publication No: US-2012-0071468-A1), andPCT/US2011/048472 (PCT Publication No: WO 2012/024616)).

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method istropisetron.

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method isnimetazepam and/or analogues thereof (see, e.g., U.S. Ser. No.13/213,960 (U.S. Patent Publication No: US-2012-0071468-A1), andPCT/US2011/048472 (PCT Publication No: WO 2012/024616)).

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method is atropinol ester or related ester (see, e.g., U.S. Ser. No. 61/514,381).

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method is a TrkAkinase inhibitor (e.g., ADDN-1351) and/or analogues thereof (see, e.g.,U.S. Ser. No. 61/525,076).

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method is a D2receptor agonists and/or an alpha1-adrenergic receptor antagonists.

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method is an ASBIsas described and/or claimed in U.S. Ser. No. 61/728,688, filed on Nov.20, 2012 which is incorporated herein by reference for the active agentsdescribed herein including, but not limited to galangin, a galanginprodrug, rutin, a, and other flavonoids as described or claimed therein.

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method is one ormore cholinesterase inhibitors (e.g., acetyl- and/orbutyrylchlolinesterase inhibitors).

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method is one ormore muscarinic antagonists (e.g., m₁ agonists or m₂ antagonists).

Certain embodiments provide a pharmaceutical composition comprising aneffective amount of one or more hydantoins described herein and anadditional therapeutic agent, and/or a method of treatment orprophylaxis comprising administration of one or more hydantoinsdescribed herein in conjunction with an additional therapeutic agentwhere the therapeutic agent in the formulation and/or method is one ormore compounds selected from the group consisting of cholinesteraseinhibitors (such as, for example,(.+−.)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]methy-1]-1H-inden-1-onehydrochloride, i.e, donepezil hydrochloride, available as the ARICEPT®brand of donepezil hydrochloride), N-methyl-D-aspartate receptorinhibitors (such as, for example, Namenda® (memantine HCl));anti-amyloid antibodies (such as bapineuzumab, Wyeth/Elan), gammasecretase inhibitors, gamma secretase modulators, and beta secretaseinhibitors other than the hydantoins described herein.

Additional Indications.

Use of Hydantoin in Age Related Macular Degeneration and Glaucoma.

While in various embodiments, the use of APP-Binding-BACE Inhibitors(ABBIs), e.g., the various hydantoins described herein, are contemplatedfor the preventing or delaying the onset of a pre-Alzheimer's conditionand/or cognitive dysfunction, and/or ameliorating one or more symptomsof a pre-Alzheimer's condition and/or cognitive dysfunction, orpreventing or delaying the progression of a pre-Alzheimer's condition orcognitive dysfunction to Alzheimer's disease, and/or for the treatmentof Alzheimer's disease, other uses of ABBIs are also contemplated. Inparticular, in certain embodiments, the use of ABBIs is contemplated forthe treatment and/or prophylaxis of age-related macular degenerationand/or glaucoma.

Without being bound to a particular theory, it is believed that abnormalextracellular deposition of proteins may contribute to age-relatedmacular degeneration (AMD) pathogenesis and progression, which is alsothe case in Alzheimer's disease and atherosclerosis. In both conditions,the protein deposits contain many shared constituents such as apoE,complement, and Aβ peptides. For instance, in human AMD, Aβ peptidedeposition is associated with drusen, where it accumulates andcolocalizes with activated complement components (Anderson et al. (2004)Exp. Eye. Res., 78:243-256; Dentchev et al. (2003)Mol. Vis., 9: 184-190;Johnson et al. (2002) Proc Natl Acad Sci USA 99: 11830-11835.). Luibl etal. (2006) J. Clin. Invest., 116: 378-385, showed the presence ofpotentially toxic amyloid oligomers in drusen, sub-RPE basal deposits,and RPE of human donor eyes using an antibody that specificallyrecognizes the oligomeric form of Aβ. These Aβ oligomers were notdetected in control age-matched donor eyes without drusen. Isas et al.(2010) Invest. Ophthalmol Vis. Sci., 51: 1304-1310, also detectedsoluble as well as mature Aβ fibrils in drusen. Collectively, thesefindings implicate Aβ in the pathogenesis of AMD. In addition, Aβpeptide has been detected in sub-RPE basal deposits and neovascularlesions in a murine model of AMD (Ding et al. (2008) Vision Res., 48:339-345; Malek et al. (2005) Proc Natl Acad Sci USA, 102: 11900-11905).In this model, aged human APOE4-targeted replacement mice (APOE4 mice)fed a high-fat, cholesterol-enriched (HFC) diet (APOE4-HFC mice) exhibitmorphologic hallmarks observed in both dry and wet AMD. These hallmarksinclude thick diffuse sub-RPE deposits, lipid- and protein-containingfocal drusen-like deposits, thickening of Bruch's membrane, patchyregions of RPE atrophy opposed to areas of photoreceptor degeneration,and CNV (Malek et al. (2005) Proc Natl Acad Sci USA, 102: 11900-11905).It is believed that, in the APOE4-HFC mouse model of AMD, Aβaccumulation provokes damage at the level of the RPE/choroid and haspreviously been shown that systemic administration of anti-Aβ40-specificantibodies can partially attenuate the decline in visual functionexhibited in this model (Ding et al. (2008) Vision Res., 48: 339-345).It has also been demonstrated that anti-Aβ immunotherapy simultaneouslytargeting both Aβ40 and Aβ42 blocks histopathologic changes andcompletely protects visual function in APOE4-HFC mice (Ding et al.(2011) Proc. Nat'l. Acad. Sci. U.S.A., 108(28): ε279-ε287).

Without being bound by a particular theory, it is believed that APPprocessing to A3 in the eye occurs by the activities of BACE andγ-secretase in the retina and retinal pigmented epithelial (RPE) celllayers and that sAPPc and A3 are secreted into the vitreous humor (see,e.g., (Prakasam et al. (2008) J. Alzh. Dis., 20: 1243-1253). A3 isfurther transported into the aqueous humor where it is readily measured.

In view of these findings, it is believe that ABBIs, e.g., thehydantoins described herein, can find use in the treatment orprophylaxis of age-related macular degeneration (AMD) and/or glaucoma.Accordingly, it is believed that ABBIs can be administered to a subjectto slow or prevent the appearance of AMD (and/or glaucoma), and/or toreduce one or more symptoms of AMD, and/or to slow, stop, or reverseprogression of the disease. In various embodiments one or more ABBIs(e.g., any one or more of the active agent(s) described herein) areadministered to a subject (e.g., a human, a non-human mammal) for thesepurposes. As described above, in various embodiments, the ABBI isadministered via a route selected from the group consisting of oraldelivery, isophoretic delivery, transdermal delivery, parenteraldelivery, aerosol administration, administration via inhalation,intravenous administration, and rectal administration.

In certain embodiments, the administration is directly to the eye. Thusfor example, in certain embodiments, the agent(s) can be administered tothe eye in the form of eye drops, via intraocular injection, and thelike.

Typically the ABBIs are administered in an effective amount for thetreatment and/or prophylaxis of AMD or glaucoma, where the effectiveamount will vary by the modality of administration. In certainembodiments effective amount is an amount sufficient to mitigating in amammal one or more symptoms associated with age-related maculardegeneration (AMD). In certain embodiments the effective amount is anamount, an amount sufficient to reduce the risk or delaying the onset,and/or reduce the ultimate severity of a AMD disease (or glaucoma)characterized by reduction of A3 in the vitreous and/or aqueous humorand/or the amyloid deposits on the retina and/or the RPE cell layer.

Assay Systems to Evaluate APP Processing

Without being bound to a particular theory, it is believed that theactive agent(s) described herein (e.g., ABBIs such as the hydantoinsdescribed herein) promote processing of APP by the nonamyloidogenicpathway and/or reduce or inhibits processing of APP by the amyloidogenicpathway. In the nonamyloidogeic pathway, APP is first cleaved byα-secretase within the Aβ sequence, releasing the APPsα ectodomain(“sAPPα”). In contrast, the amyloidogenic pathway is initiated whenβ-secretase cleaves APP at the amino terminus of the Aβ, therebyreleasing the APPsβ ectodomain (“sAPPYβ”). APP processing by thenonamyloidogenic and amyloidogenic pathways is known in the art andreviewed, e.g., by Xu (2009) J Alzheimers Dis., 16(2): 211-224, and DeStrooper, et al. (2010 Nat Rev Neurol 6(2): 99-107.

One method to evaluate the efficacy of the active agent(s) is todetermine a reduction or elimination in the level of APP processing bythe amyloidogenic pathway, e.g., a reduction or elimination in the levelof APP processing by β-secretase cleavage in response to theadministration of the agent(s) of interest. Assays for determining theextent of APP cleavage at the β-secretase cleavage site are well knownin the art. Illustrative assays are described, for example, in U.S. Pat.Nos. 5,744,346 and 5,942,400. Kits for determining the presence andlevels in a biological sample of sAPPα and sAPPβ, as well as APPneo andAβ commercially available, e.g., from PerkinElmer.

ABBI Assay.

APP Binding BACE Inhibitor (ABBI) activity of any of the compoundsdescribed herein can readily be verified using, for example, assaysdescribed herein. Basically, in certain embodiments a pair the assaysare utilized to identify ABBI compounds that inhibit BACE cleavage ofthe MBP-C125 APP substrate, resulting in the inhibition of theproduction of C99 and the β-site peptide substrate (P5-P5′) and alsointeracts with APP, e.g., as measured by surface plasmon resonance (SPR)analysis.

In one illustrative embodiment, an MBP-C125 APP695 wt fusion protein canbe used as one of the substrates and the second substrate can be thecommercially available P5-P5′ fluorescence substrate. Each of thesesubstrates is incubated with recombinant BACE (R&D (cat#931-AS-050) in,for example, a 96 well plate format. For the MBP-C125 substrate the C-99product from the BACE cleavage can be measured using an AlphaLisa assayas a readout. For the P5-5′ substrate the loss of fluorescence upon BACEcleavage can be used as the readout. For the SPR assay the bindinganalysis of the hydantoins to fragments of the ectodomain of APP (eAPP)that are recombinantly prepared (Libeu et al. (2012) PLoS ONE 7(6):e40027) would be done. An ABBI would inhibit the BACE cleavage of theMBP-C125 and/or the fluorescence substrate and would also bind to theectodomain of APP such as the APP₂₃₀₋₆₂₄ fragment.

Other Cell Free Assays

Illustrative assays that can be used to demonstrate the inhibitoryactivity of the active agent(s) are described, for example, in WO2000/017369, WO 2000/0003819, and U.S. Pat. Nos. 5,942,400 and5,744,346. Such assays can be performed in cell-free incubations or incellular incubations using cells expressing an alpha-secretase and/orbeta-secretase and an APP substrate having a alpha-secretase andbeta-secretase cleavage sites.

In one illustrative embodiment, the agent(s) of interest are contactedwith an APP substrate containing alpha-secretase and beta-secretasecleavage sites of APP, for example, a complete APP or variant, an APPfragment, or a recombinant or synthetic APP substrate containing theamino acid sequence: KM-DA or NL-DA (APP-SW), is incubated in thepresence of an alpha-secretase and/or beta-secretase enzyme, a fragmentthereof, or a synthetic or recombinant polypeptide variant havingalpha-secretase or beta-secretase activity and effective to cleave thealpha-secretase or beta-secretase cleavage sites of APP, underincubation conditions suitable for the cleavage activity of the enzyme.Agent(s) having the desired activity reduce or prevent cleavage of theAPP substrate. Suitable substrates optionally include derivatives thatmay be fusion proteins or peptides that contain the substrate peptideand a modification useful to facilitate the purification or detection ofthe peptide or its alpha-secretase and/or beta-secretase cleavageproducts. Useful modifications include the insertion of a knownantigenic epitope for antibody binding; the linking of a label ordetectable moiety, the linking of a binding substrate, and the like.

Suitable incubation conditions for a cell-free in vitro assay include,for example: approximately 200 nanomolar to 10 micromolar substrate,approximately 10 to 200 picomolar enzyme, and approximately 0.1nanomolar to 10 micromolar of the agent(s), in aqueous solution, at anapproximate pH of 4-7, at approximately 37° C., for a time period ofapproximately 10 minutes to 3 hours. These incubation conditions areillustrative only, and can be varied as required for the particularassay components and/or desired measurement system. Optimization of theincubation conditions for the particular assay components should accountfor the specific alpha-secretase and/or beta-secretase enzyme used andits pH optimum, any additional enzymes and/or markers that might be usedin the assay, and the like. Such optimization is routine and will notrequire undue experimentation.

Another illustrative assay utilizes a fusion peptide having maltosebinding protein (MBP) fused to the C-terminal 125 amino acids of APP-SW.The MBP portion is captured on an assay substrate by anti-MBP captureantibody. Incubation of the captured fusion protein in the presence ofalpha-secretase and/or beta-secretase results in cleavage of thesubstrate at the alpha-secretase and/or beta-secretase cleavage sites,respectively. This system can be used to screen for the inhibitoryactivity of the agent(s) of interest. Analysis of the cleavage activitycan be, for example, by immunoassay of cleavage products. One suchimmunoassay detects a unique epitope exposed at the carboxy terminus ofthe cleaved fusion protein, for example, using the antibody SW192. Thisassay is described, for example, in U.S. Pat. No. 5,942,400.

Cellular Assays

Numerous cell-based assays can be used to evaluate the activity ofagent(s) of interest on relative alpha-secretase activity tobeta-secretase activity and/or processing of APP to releaseamyloidogenic versus non-amyloidogenic Aβ oligomers. Contact of an APPsubstrate with an alpha-secretase and/or beta-secretase enzyme withinthe cell and in the presence or absence of the agent(s) can be used todemonstrate alpha-secretase promoting and/or beta-secretase inhibitoryactivity of the agent(s). Preferably, the assay in the presence of theagent(s) provides at least about 30%, most preferably at least about 50%inhibition of the enzymatic activity, as compared with a non-inhibitedcontrol.

In one embodiment, cells that naturally express alpha-secretase and/orbeta-secretase are used. Alternatively, cells are modified to express arecombinant alpha-secretase and/or beta-secretase or synthetic variantenzymes, as discussed above. The APP substrate may be added to theculture medium and is preferably expressed in the cells. Cells thatnaturally express APP, variant or mutant forms of APP, or cellstransformed to express an isoform of APP, mutant or variant APP,recombinant or synthetic APP, APP fragment, or synthetic APP peptide orfusion protein containing the alpha-secretase and/or beta-secretase APPcleavage sites can be used, provided that the expressed APP is permittedto contact the enzyme and enzymatic cleavage activity can be analyzed.

Human cell lines that normally process Aβ from APP provide a usefulmeans to assay inhibitory activities of the agent(s). Production andrelease of Aβ and/or other cleavage products into the culture medium canbe measured, for example by immunoassay, such as Western blot orenzyme-linked immunoassay (EIA) such as by ELISA.

Cells expressing an APP substrate and an active alpha-secretase and/orbeta-secretase can be incubated in the presence of the agents todemonstrate relative enzymatic activity of the alpha-secretase and/orbeta-secretase as compared with a control. Relative activity of thealpha-secretase to the beta-secretase can be measured by analysis of oneor more cleavage products of the APP substrate. For example, inhibitionof beta-secretase activity against the substrate APP would be expectedto decrease release of specific beta-secretase induced APP cleavageproducts such as Aβ (e.g., Aβ40 or Aβ42), sAPPβ and APPneo. Promotion orenhancement of alpha-secretase activity against the substrate APP wouldbe expected to increase release of specific alpha-secretase induced APPcleavage products such as sAPPα and p3 peptide.

Although both neural and non-neural cells process and release Aβ, levelsof endogenous beta-secretase activity are low and often difficult todetect by EIA. The use of cell types known to have enhancedbeta-secretase activity, enhanced processing of APP to Aβ, and/orenhanced production of Aβ are therefore preferred. For example,transfection of cells with the Swedish Mutant form of APP (APP-SW); withthe Indiana Mutant form (APP-IN); or with APP—SW-IN provides cellshaving enhanced beta-secretase activity and producing amounts of Aβ thatcan be readily measured.

In such assays, for example, the cells expressing APP, alpha-secretaseand/or beta-secretase are incubated in a culture medium under conditionssuitable for alpha-secretase and/or beta-secretase enzymatic activity atits cleavage site on the APP substrate. On exposure of the cells to theagent(s), the amount of Aβ released into the medium and/or the amount ofCTF99 fragments of APP in the cell lysates is reduced as compared withthe control. The cleavage products of APP can be analyzed, for example,by immune reactions with specific antibodies, as discussed above.

In certain embodiments, preferred cells for analysis of alpha-secretaseand/or beta-secretase activity include primary human neuronal cells,primary transgenic animal neuronal cells where the transgene is APP, andother cells such as those of a stable 293 cell line expressing APP, forexample, APP-SW.

In vivo Assays: Animal Models

Various animal models can be used to analyze the activity of agent(s) ofinterest on relative alpha-secretase and/or beta-secretase activityand/or processing of APP to release Aβ. For example, transgenic animalsexpressing APP substrate, alpha-secretase and/or beta-secretase enzymecan be used to demonstrate inhibitory activity of the agent(s). Certaintransgenic animal models have been described, for example, in U.S. Pat.Nos. 5,877,399; 5,612,486; 5,387,742; 5,720,936; 5,850,003; 5,877,015,and 5,811,633, and in Ganes et al. (1995) Nature 373: 523. Preferred areanimals that exhibit characteristics associated with the pathophysiologyof AD. Administration of the agent(s) to the transgenic mice describedherein provides an alternative method for demonstrating the inhibitoryactivity of the agent(s). Administration of the agent(s) in apharmaceutically effective carrier and via an administrative route thatreaches the target tissue in an appropriate therapeutic amount is alsopreferred.

Inhibition of beta-secretase mediated cleavage of APP at thebeta-secretase cleavage site and of Aβ release can be analyzed in theseanimals by measure of cleavage fragments in the animal's body fluidssuch as cerebral fluid or tissues. Likewise, promotion or enhancement ofalpha-secretase mediated cleavage of APP at the alpha-secretase cleavagesite and of release of sAPPα can be analyzed in these animals by measureof cleavage fragments in the animal's body fluids such as cerebral fluidor tissues. In certain embodiments, analysis of brain tissues for Aβdeposits or plaques is preferred.

On contacting an APP substrate with an alpha-secretase and/orbeta-secretase enzyme in the presence of the agent(s) under conditionssufficient to permit enzymatic mediated cleavage of APP and/or releaseof Aβ from the substrate, desirable agent(s) are effective to reducebeta-secretase-mediated cleavage of APP at the beta-secretase cleavagesite and/or effective to reduce released amounts of Aβ. The agent(s) arealso preferably effective to enhance alpha-secretase-mediated cleavageof APP at the alpha-secretase cleavage site and to increase releasedamounts of sAPPα. Where such contacting is the administration of theagent(s) to an animal model, for example, as described above, theagent(s) is effective to reduce A3 deposition in brain tissues of theanimal, and to reduce the number and/or size of beta amyloid plaques.Where such administration is to a human subject, the agent(s) iseffective to inhibit or slow the progression of disease characterized byenhanced amounts of A3, to slow the progression of AD in the, and/or toprevent onset or development of AD in a patient at risk for the disease.

Methods of Monitoring Clinical Efficacy

In various embodiments, the effectiveness of treatment can be determinedby comparing a baseline measure of a parameter of disease beforeadministration of the agent(s) (e.g., hydantoins described herein, or atautomer(s) or stereoisomer(s) thereof, or pharmaceutically acceptablesalts or solvates of said hydantoin(s), said stereoisomer(s), or saidtautomer(s), or analogues, derivatives, or prodrugs thereof) iscommenced to the same parameter one or more time points after theagent(s) or analog has been administered. One illustrative parameterthat can be measured is a biomarker (e.g., a peptide oligomer) of APPprocessing. Such biomarkers include, but are not limited to increasedlevels of sAPPα, p3 (Aβ17-42 or Aβ17-40), sAPPβ, soluble Aβ40, and/orsoluble Aβ42 in the blood, plasma, serum, urine, mucous or cerebrospinalfluid (CSF). Detection of increased levels of sAPPα and/or p3, anddecreased levels of sAPPβ and/or APPneo is an indicator that thetreatment is effective. Conversely, detection of decreased levels ofsAPPα and/or p3, and/or increased levels of sAPPβ, APPneo, Tau orphospho-Tau (pTau) is an indicator that the treatment is not effective.

Another parameter to determine effectiveness of treatment is the levelof amyloid plaque deposits in the brain. Amyloid plaques can bedetermined using any method known in the art, e.g., as determined by CT,PET, PIB-PET and/or MRI. Administration of the agent(s)) (e.g.,hydantoins described herein, or a tautomer(s) or stereoisomer(s)thereof, or pharmaceutically acceptable salts or solvates of saidhydantoin(s), said stereoisomer(s), or said tautomer(s), or analogues,derivatives, or prodrugs thereof) can result in a reduction in the rateof plaque formation, and even a retraction or reduction of plaquedeposits in the brain. Effectiveness of treatment can also be determinedby observing a stabilization and/or improvement of cognitive abilitiesof the subject. Cognitive abilities can be evaluated using anyart-accepted method, including for example, Clinical Dementia Rating(CDR), the mini-mental state examination (MMSE) or Folstein test,evaluative criteria listed in the DSM-IV (Diagnostic and StatisticalManual of Mental Disorders, Fourth Edition) or DSM-V, and the like.

Clinical efficacy can be monitored using any method known in the art.Measurable biomarkers to monitor efficacy include, but are not limitedto, monitoring blood, plasma, serum, urine, mucous or cerebrospinalfluid (CSF) levels of sAPPα, sAPPβ, Aβ42, Aβ40, APPneo and p3 (e.g.,Aβ17-42 or Aβ17-40). Detection of increased levels of sAPPα and/or p3,and decreased levels of sAPPβ and/or APPneo are indicators that thetreatment or prevention regime is efficacious. Conversely, detection ofdecreased levels of sAPPα and/or p3, and increased levels of sAPPβand/or APPneo are indicators that the treatment or prevention regime isnot efficacious. Other biomarkers include Tau and phospho-Tau (pTau).Detection of decreased levels of Tau and pTau are indicators that thetreatment or prevention regime is efficacious.

Efficacy can also be determined by measuring amyloid plaque load in thebrain. The treatment or prevention regime is considered efficacious whenthe amyloid plaque load in the brain does not increase or is reduced.Conversely, the treatment or prevention regime is consideredinefficacious when the amyloid plaque load in the brain increases.Amyloid plaque load can be determined using any method known in the art,e.g., including CT, PET, PIB-PET and/or MRI.

Efficacy can also be determined by measuring the cognitive abilities ofthe subject. Cognitive abilities can be measured using any method knownin the art. Illustrative tests include assigning a Clinical DementiaRating (CDR) score or applying the mini mental state examination (MMSE)(Folstein, et al., Journal of Psychiatric Research 12 (3): 189-98).Subjects who maintain the same score or who achieve an improved score,e.g., when applying the CDR or MMSE, indicate that the treatment orprevention regime is efficacious. Conversely, subjects who receive ascore indicating diminished cognitive abilities, e.g., when applying theCDR or MMSE, indicate that the treatment or prevention regime has notbeen efficacious.

In certain embodiments, the monitoring methods can entail determining abaseline value of a measurable biomarker or parameter (e.g., amyloidplaque load or cognitive abilities) in a subject before administering adosage of the agent(s), and comparing this with a value for the samemeasurable biomarker or parameter after treatment.

In other methods, a control value (e.g., a mean and standard deviation)of the measurable biomarker or parameter is determined for a controlpopulation. In certain embodiments, the individuals in the controlpopulation have not received prior treatment and do not have AD, MCI,nor are at risk of developing AD or MCI. In such cases, if the value ofthe measurable biomarker or clinical parameter approaches the controlvalue, then treatment is considered efficacious. In other embodiments,the individuals in the control population have not received priortreatment and have been diagnosed with AD or MCI. In such cases, if thevalue of the measurable biomarker or clinical parameter approaches thecontrol value, then treatment is considered inefficacious.

In other methods, a subject who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for one ormore of the biomarkers or clinical parameters to determine whether aresumption of treatment is required. The measured value of one or moreof the biomarkers or clinical parameters in the subject can be comparedwith a value previously achieved in the subject after a previous courseof treatment. Alternatively, the value measured in the subject can becompared with a control value (mean plus standard deviation/ANOVA)determined in population of subjects after undergoing a course oftreatment. Alternatively, the measured value in the subject can becompared with a control value in populations of prophylactically treatedsubj ects who remain free of symptoms of disease, or populations oftherapeutically treated subjects who show amelioration of diseasecharacteristics. In such cases, if the value of the measurable biomarkeror clinical parameter approaches the control value, then treatment isconsidered efficacious and need not be resumed. In all of these cases, asignificant difference relative to the control level (e.g., more than astandard deviation) is an indicator that treatment should be resumed inthe subject.

In certain embodiments the tissue sample for analysis is typicallyblood, plasma, serum, urine, mucous or cerebrospinal fluid from thesubject.

Kits.

In various embodiments, the active agent(s) (e.g., hydantoins) describedherein thereof can be enclosed in multiple or single dose containers.The enclosed agent(s) can be provided in kits, for example, includingcomponent parts that can be assembled for use. For example, an activeagent in lyophilized form and a suitable diluent may be provided asseparated components for combination prior to use. A kit may include anactive agent and a second therapeutic agent for co-administration. Theactive agent and second therapeutic agent may be provided as separatecomponent parts. A kit may include a plurality of containers, eachcontainer holding one or more unit dose of the compounds. The containersare preferably adapted for the desired mode of administration,including, but not limited to tablets, gel capsules, sustained-releasecapsules, and the like for oral administration; depot products,pre-filled syringes, ampules, vials, and the like for parenteraladministration; and patches, medipads, creams, and the like for topicaladministration, e.g., as described herein.

In certain embodiments, a kit is provided where the kit comprises one ormore hydantoin compounds described herein, or a tautomer or stereoisomerthereof, or pharmaceutically acceptable salt or solvate of saidcompound, said stereoisomer, or said tautomer, preferably provided as apharmaceutical composition and in a suitable container or containersand/or with suitable packaging; optionally one or more additional activeagents, which if present are preferably provided as a pharmaceuticalcomposition and in a suitable container or containers and/or withsuitable packaging; and optionally instructions for use, for examplewritten instructions on how to administer the compound or compositions.

In another embodiment, a kit is provided that comprises a singlecontainer or multiple containers: (a) a pharmaceutically acceptablecomposition comprising one or more compounds of claim 1 and/or any ofcompounds 1-10 shown in FIGS. 1 and 2, or a tautomer or stereoisomerthereof, or pharmaceutically acceptable salt or solvate of saidcompound, said stereoisomer, or said tautomer, optionally apharmaceutically acceptable composition comprising one or moreadditional therapeutic agents; and optionally instructions for use theiruse. The kit may optionally comprise labeling (e.g., instructionalmaterials) appropriate to the intended use or uses.

As with any pharmaceutical product, the packaging material(s) and/orcontainer(s) are designed to protect the stability of the product duringstorage and shipment. In addition, the kits can include instructions foruse or other informational material that can advise the user such as,for example, a physician, technician or patient, regarding how toproperly administer the composition(s) as prophylactic, therapeutic, orameliorative treatment of the disease of concern. In some embodiments,instructions can indicate or suggest a dosing regimen that includes, butis not limited to, actual doses and monitoring procedures.

In some embodiments, the instructions can include informational materialindicating that the administering of the compositions can result inadverse reactions including but not limited to allergic reactions suchas, for example, anaphylaxis. The informational material can indicatethat allergic reactions may exhibit only as mild pruritic rashes or maybe severe and include erythroderma, vasculitis, anaphylaxis,Steven-Johnson syndrome, and the like. In certain embodiments theinformational material(s) may indicate that anaphylaxis can be fatal andmay occur when any foreign protein is introduced into the body. Incertain embodiments the informational material may indicate that theseallergic reactions can manifest themselves as urticaria or a rash anddevelop into lethal systemic reactions and can occur soon after exposuresuch as, for example, within 10 minutes. The informational material canfurther indicate that an allergic reaction may cause a subject toexperience paresthesia, hypotension, laryngeal edema, mental statuschanges, facial or pharyngeal angioedema, airway obstruction,bronchospasm, urticaria and pruritus, serum sickness, arthritis,allergic nephritis, glomerulonephritis, temporal arthritis,eosinophilia, or a combination thereof.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedherein. Such media include, but are not limited to electronic storagemedia (e.g., magnetic discs, tapes, cartridges, chips), optical media(e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

In some embodiments, the kits can comprise one or more packagingmaterials such as, for example, a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (I.V.) bag, envelope, and the like;and at least one unit dosage form of an agent comprising active agent(s)described herein and a packaging material. In some embodiments, the kitsalso include instructions for using the composition as prophylactic,therapeutic, or ameliorative treatment for the disease of concern.

In some embodiments, the articles of manufacture can comprise one ormore packaging materials such as, for example, a box, bottle, tube,vial, container, sprayer, insufflator, intravenous (I.V.) bag, envelope,and the like; and a first composition comprising at least one unitdosage form of an agent comprising one or more hydantoins describedherein, or a tautomer(s) or stereoisomer(s) thereof, or pharmaceuticallyacceptable salts or solvates of said hydantoin(s), said stereoisomer(s),or said tautomer(s), or analogues, derivatives, or prodrugs thereofwithin the packaging material, along with a second compositioncomprising a second agent such as, for example, an agent used in thetreatment and/or prophylaxis of Alzheimer's disease (e.g., as describedherein), or any prodrugs, codrugs, metabolites, analogs, homologues,congeners, derivatives, salts and combinations thereof. In someembodiments, the articles of manufacture may also include instructionsfor using the composition as a prophylactic, therapeutic, orameliorative treatment for the disease of concern.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Synthesis of Compound15-(3,5-difluorophenyl)-5-phenylimidazolidine-2,4-dione (Hydantoin-1)Step 1: Synthesis of(3,5-difluorophenyl)(2-phenyl-1,3-dithian-2-yl)methanol

2-Phenyl-1,3-dithiane (1.791 g, 9.12 mmol) was dissolved in 20 ml of dryTHF and cooled to 0° C. BuLi (6.84 ml, 10.94 mmol) was added dropwiseunder nitrogen and the mixture was stirred for 30 min at 0° C. Asolution of 3,5-difluorobenzaldehyde (1.00 ml, 9.12 mmol) in THF (10 ml)was added and the mixture was stirred for 30 minutes, then warmed toambient temperature over 1 hour and quenched with saturated ammoniumchloride solution. The organic phase was washed with brine and driedwith sodium sulfate. The solvent was removed in vacuo to give crude(3,5-difluorophenyl)(2-phenyl-1,3-dithian-2-yl)methanol (3.15 g, 9.31mmol, 104% yield) as a thick yellow oil. The residue was carried throughto the next step.

Step 2: Synthesis of 2-(3,5-difluorophenyl)-2-hydroxy-1-phenylethanone

(3,5-Difluorophenyl)(2-phenyl-1,3-dithian-2-yl)methanol (3.15 g, 9.31mmol) was dissolved in 15 ml of acetonitrile and 2.5 ml of water.

Bis(trifluoroacetoxy)iodobenzene (5.00 g, 11.63 mmol) in 10 ml ofacetonitrile was slowly added at ambient temperature to the vigorouslystirred solution. After 30 minutes TLC (25% EtOAC/hexane) analysisindicated a complete reaction. EtOAc (150 ml) was added and the mixturewas rinsed with saturated sodium bicarbonate solution (50 ml) and brine(50 ml). The organic fractions were dried, and the solvent was removedin vacuo. The crude product was purified by flash column chromatography(12.5% EtOAc/hexane) to give2-(3,5-difluorophenyl)-2-hydroxy-1-phenylethanone (1.10 g, 4.43 mmol,48%) as a pale yellow solid. The proton NMR was consistent with theproposed structure.

Step 3: Synthesis of 1-(3,5-difluorophenyl)-2-phenylethane-1,2-dione

2-(3,5-difluorophenyl)-2-hydroxy-1-phenylethanone (1.10 g, 4.43 mmol)was dissolved in 80% acetic acid together with diacetoxycopper hydrate(44 mg, 0.22 mmol) and ammonium nitrate (0.30 g, 3.75 mmol). The mixturewas refluxed for 2.5 hours and then cooled. The reaction mixture waspoured into ethyl acetate (50 ml) and washed with brine (2×25 ml), driedover sodium sulfate, filtered and evaporated. The crude material waspurified by column chromatography (5% EtOAc/hexane) to give1-(3,5-difluorophenyl)-2-phenylethane-1,2-dione (1.10 g, 4.43 mmol,quant.) as a bright yellow solid. The proton NMR was consistent with theproposed structure.

Step 4: Synthesis of5-(3,5-difluorophenyl)-5-phenylimidazolidine-2,4-dione

To a solution of 1-(3,5-difluorophenyl)-2-phenylethane-1,2-dione (0.99g, 4.02 mmol), urea (0.435 g, 7.24 mmol) in ethanol (20 ml) and water (5ml) was added solid NaOH (0.29 g, 7.24 mmol). The reaction mixture wasrefluxed until TLC (50% EtOAc/hexane) analysis indicated a completereaction. The reaction mixture was diluted with water (30 ml) andcarefully acidified with 2M HCl to pH 5. The reaction mixture wasextracted with ethyl acetate (100 ml) and washed with water (50 ml) andbrine (50 ml). The organic extract was dried over sodium sulfate,filtered and evaporated to give a residue that was triturated withacetone and hexane mixtures to afford5-(3,5-difluorophenyl)-5-phenylimidazolidine-2,4-dione (0.220 g) as asolid that was highly hydrated with water as judged by NMR spectroscopy.The solid, after heating (120° C.) under vacuum overnight afforded thedesired product (0.20 g, 0.69 mmol, 17%) as a white powder. ¹H NMR (400MHz, d₆-DMSO) δ ppm 11.31 (brs, 1H), 9.44 (s, 1H), 7.37 (m, 6H), 7.10(d, J 6.77 Hz, 2H); ¹³C NMR (100 MHz, d₆-DMSO) δ ppm 173.89, 163.52,163.39, 161.06, 160.93, 155.78, 143.79, 143.70, 143.61, 139.16, 128.82,128.44, 126.26, 110.12, 110.04, 109.93, 109.85, 104.11, 103.86, 103.60,69.43 (note: C—F coupling was observed in several instances giving riseto doublet and triplet signals; LC (220 nm): R_(t)=4.09 min, LC purity:95.8%, m/z (M-1): 300.3.

Example 2 Synthesis of FAH-2:2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-oneStep 1: Synthesis of 2-(3,5-difluorophenyl)-1,3-dithiane

BF₃.OMe₂ (0.70 ml, 7.62 mmol) was added dropwise to a solution of1,3-propanedithiol (0.90 ml, 8.94 mmol) and 3,5-difluorobenzaldehyde(1.00 ml, 8.94 mmol) in DCM (50 ml) at 0° C. The reaction was stirred atambient temperature for 1 hour where TLC (5% EtOAc/hexane) indicated acomplete reaction. The reaction mixture was then diluted with DCM (50ml), filtered through Celite (and the Celite pad was washed withadditional DCM (3×20 ml) and the filtrate washed with brine (50 ml),saturated NaHCO₃ (3×50 ml), 10% KOH solution (50 ml), water (50 ml) andbrine (50 ml) and finally dried over sodium sulfate. The organic extractwas filtered and evaporated to afford2-(3,5-difluorophenyl)-1,3-dithiane (2.14 g, 9.21 mmol, 103%) as whitecrystalline needles. The proton NMR was consistent with the proposedstructure.

Step 2: Synthesis of(4-(difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol

2-(3,5-Difluorophenyl)-1,3-dithiane (2.14 g, 9.21 mmol) was dissolved in20 ml of dry THF and cooled to 0° C. BuLi (8.50 ml, 10.20 mmol) wasadded dropwise under nitrogen and the mixture was stirred for 15 min at0° C. A solution of 4-(difluoromethoxy)benzaldehyde (1.30 ml, 9.33 mmol)in THF (10 ml) was added and the mixture was stirred for 10 minutes,then warmed to ambient temperature over 10 minutes and quenched withsaturated ammonium chloride solution. The organic phase was washed withbrine and dried with sodium sulfate. The solvent was removed in vacuo togive a residue that was purified by flash column chromatography (10%EtOAc/hexane) to afford(4-(difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol(1.90 g, 4.70 mmol, 51%) as a thick yellow oil. The proton NMR wasconsistent with the proposed structure.

Step 3: Synthesis of2-(4-(difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone

(4-(Difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol(1.90 g, 4.70 mmol) was dissolved in 15 ml acetonitrile and 2.5 ml ofwater. Bis(trifluoroacetoxy)iodobenzene (2.53 g, 5.87 mmol) in 10 ml ofacetonitrile was slowly added at ambient temperature to the vigorouslystirred solution. After 30 minutes TLC (25% EtOAC/hexane) analysisindicated a complete reaction. EtOAc (150 ml) was added and the mixturewas rinsed with saturated sodium bicarbonate solution (50 ml) and brine(50 ml). The organic fractions were dried, and the solvent was removedin vacuo. The crude product was purified by flash column chromatography(12.5% EtOAc/hexane) to give2-(4-(difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone(0.460 g, 1.46 mmol, 31%) as a pale yellow solid. The proton NMR wasconsistent with the proposed structure.

Step 4: Synthesis of1-(4-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione

2-(4-(Difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone(0.46 g, 1.46 mmol) was dissolved in 80% acetic acid together withdiacetoxycopper hydrate (26 mg, 0.13 mmol) and ammonium nitrate (0.18 g,2.25 mmol). The mixture was refluxed for 90 minutes and then cooled. Thereaction mixture was poured into ethyl acetate (50 ml) and washed withbrine (2×25 ml), dried over sodium sulfate, filtered and evaporated. Thecrude material was passed through a silica-gel plug, evaporated andazeotroped with toluene (3×20 ml) to remove excess acetic acid to givecrude1-(4-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione(0.456 g, 1.46 mmol, 100%) as a bright yellow solid. The crude solid wascarried through to the next step.

Step 5: Synthesis of2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one

1-(4-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione(0.456 g, 1.46 mmol) in ethanol (20 ml) and water (5 ml) was added1-methylguanidine hydrochloride (0.16 g, 1.46 mmol) and potassiumcarbonate (0.61 g, 4.38 mmol). The mixture was allowed to reflux for 3hours and then cooled to ambient temperature. The volatiles were removedin vacuo and the residue was taken up in water and extracted intochloroform (50 ml). The organic fractions were dried with sodium sulfateand the solvent was removed in vacuo. The crude material was purified bycolumn chromatography (EtOAc) to afford2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one(0.172 g, 0.47 mmol) as a glass that was heavily contaminated with ethylacetate as judged by NMR analysis. The glass was taken into dry ethanol(3 ml) and layered with hexane (1 ml) to get a turbid solution. Thesolution was rotary evaporated to give an oil that solidified onstanding. This was dried overnight and the weight obtained was 0.150 g.The solid contained ethanol and hexane solvent residues as judged by NMRanalysis. Therefore, the solids were re-dissolved into iso-propanol,rotary evaporated and dried under high vacuum at 90° C. overnight toafford2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one(0.130 g, 0.35 mmol, 24%) as an off-white solid. ¹H NMR (400 MHz, CDCl₃)δ ppm 7.47 (d, J=8.79 Hz, 2H), 7.10-7.00 (m, 4H), 6.70 (tt, J=8.73, 2.32Hz, 1H), 6.53 (t, J_(H-F)=73.76 Hz, 1H), 5.45 (brs, 1H), 3.11 (s, 3H);¹³C NMR (100 MHz, CDCl₃) δ ppm 178.619, 164.122, 163.996, 161.650,161.524, 155.704, 150.742, 150.714, 150.687, 145.164, 145.081, 144.991,137.735, 128.390, 119.444, 118.328, 115.743, 113.158, 110.329, 110.255,110.138, 110.065, 103.421, 103.169, 102.917, 77.203, 25.966 (note: C—Fcoupling was observed in several instances giving rise to doublet andtriplet signals); LC (260 nm): R_(t)=3.899 min, LC Purity: 96.3%, m/z(M-1): 366.

Example 3 Synthesis of FAH-3:2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-oneStep 1: Synthesis of 2-(3,5-difluorophenyl)-1,3-dithiane

BF₃.OMe (1.40 ml, 15.25 mmol) was added dropwise to a solution of1,3-propanedithiol (1.81 ml, 17.87 mmol) and 3,5-difluorobenzaldehyde(2.00 ml, 17.87 mmol) in DCM (50 ml) at 0° C. The reaction was stirredat ambient temperature for 1 hour where TLC (5% EtOAc/hexane) indicateda complete reaction. The reaction mixture was then diluted withadditional DCM (50 ml), filtered through Celite, and the Celite pad waswashed with additional DCM (3×20 ml). The filtrate was washed withsaturated NaHCO3 (3×50 ml), 10% KOH solution (2×50 ml), water (2×50 ml)and brine (50 ml) and finally dried over sodium sulfate. The organicextract was filtered and evaporated to afford2-(3,5-difluorophenyl)-1,3-dithiane (4.12 g, 17.73 mmol, 99%) as a whitesolid. The proton NMR was consistent with the proposed structure.

Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

Attempt 1:

A solution of sodium chlorodifluoroacetate (3.23 g, 21.15 mmol) and4-hydroxy-3-methylbenzaldehyde (1.44 g, 10.58 mmol), potassium carbonate(2.19 g, 15.87 mmol) in a mixture of DMF (8 ml) and water (2 ml) washeated at 100° C. for 2 hours. The reaction mixture was cooled and conc.HCl (1.5 ml) followed by water (2.1 ml). The reaction mixture wasdiluted with water (20 ml) and extracted with ethyl acetate (3×25 ml).The organic extract was washed with 10% (m/v) aqueous LiCl solution(3×25 ml), dried over sodium sulfate, filtered and evaporated to give aresidue that was flash chromatographed (15% EtOAc/hexane) to give4-(difluoromethoxy)-3-methylbenzaldehyde (0.244 g, 1.31 mmol, 12%) as abrown oil and recovered 4-hydroxy-3-methylbenzaldehyde (1.164 g, 8.55mmol, 81%) as a brown solid.

The experiment was repeated again, except with the absence of water.Briefly, the experimental is given below:

Attempt 2:

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 ml) wasadded over 3 hours to a solution of DMF (15 ml) containing potassiumcarbonate (1.77 g, 12.78 mmol) at 95° C. The reaction was allowed to agefor an additional 15 minutes and then cooled. The reaction mixture wasdiluted with water (50 ml) and extracted with ethyl acetate (3×50 ml).The organic extract was washed with 10% (m/v) aqueous LiCl solution(3×25 ml), dried over sodium sulfate, filtered and evaporated to give aresidue that was flash chromatographed (15% EtOAc/hexane) to give4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37mmol, 63%) as a yellow oil. This oil was combined with that of theprevious experiment and passed through a Pasteur pipette column elutingwith 10% EtOAC/hexane to give an oil that solidified on standing (1.315g, 7.06 mmol, 67% over the two reactions). The proton NMR was consistentwith the proposed structure.

Finally, repeating the experiment using the conditions in attempt 2, anadditional 1.4 g of the desired product was isolated.

Step 3: Synthesis of(4-(difluoromethoxy)-3-methylphenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol

2-(3,5-Difluorophenyl)-1,3-dithiane (3.12 g, 13.43 mmol) was dissolvedin 30 ml of dry THF and cooled to −10° C. BuLi (1.6M, 12.0 ml, 19.20mmol) was added dropwise under nitrogen and the mixture was stirred for15 min at −10° C. to afford a blood-red solution. A solution of4-(difluoromethoxy)-3-methylbenzaldehyde (2.50 g, 13.43 mmol) in THF (10ml) was added dropwise and the mixture was stirred for 15 minutes, thenwarmed to ambient temperature over 10 minutes and quenched withsaturated ammonium chloride solution. The organic phase was washed withbrine and dried with sodium sulfate. The solvent was removed and theresidue flash chromatographed (10% EtOAc/hexane) to give(4-(difluoromethoxy)-3-methylphenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol(3.07 g, 7.22 mmol, 55%) as a thick oil that solidified on standing. TheNMR was consistent with the proposed structure.

Step 4: Synthesis of2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone

(4-(Difluoromethoxy)-3-methylphenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol(3.07 g, 7.34 mmol) was dissolved in acetonitrile (15 ml) and water (2.5ml). Bis(trifluoroacetoxy)iodobenzene (3.94 g, 9.17 mmol) inacetonitrile (10 ml) was slowly added at ambient temperature to thevigorously stirred solution. After 20 minutes TLC (20% EtOAc/hexane)analysis indicated a complete reaction. EtOAc (150 ml) was added and themixture was rinsed with saturated sodium bicarbonate solution (50 ml)and brine (50 ml). The organic fractions were dried, and the solvent wasremoved in vacuo. The crude product was purified twice by flash columnchromatography (10% EtOAc/hexane) to give2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone(0.853 g, 2.60 mmol, 35%) as a pale yellow oil. The proton NMR wasconsistent with the proposed structure.

Step 5: Synthesis of1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione

2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone(0.853 g, 2.60 mmol) was dissolved in 80% acetic acid together withdiacetoxycopper hydrate (52 mg, 0.26 mmol) and ammonium nitrate (0.156g, 1.95 mmol). The mixture was refluxed for 90 minutes and then cooled.The reaction mixture was poured into ethyl acetate (50 ml) and washedwith brine (2×25 ml), dried over sodium sulfate, filtered andevaporated. The residue was azeotroped with toluene to remove aceticacid and the residue (0.737 g, 2.26 mmol, 87%) was used directly intothe next stage.

Step 6: Synthesis of2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one

A mixture of1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione(737 mg, 2.26 mmol) in ethanol (15 ml) and dioxane (15 ml) was added1-methylguanidine hydrochloride (990 mg, 9.04 mmol) and stirred atambient temperature for 15 minutes. Sodium carbonate (958 mg, 9.04 mmol)in water (5 ml) was added and the mixture immersed into an oil bath at85° C. and stirred for 3 hours. TLC (EtOAc) indicated a completereaction. The reaction mixture was cooled to ambient temperature andconcentrated. Purification by flash chromatography (EtOAc) afforded2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one(0.52 g, 1.36 mmol, 60%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δppm 7.34-7.24 (m, 2H), 7.08-6.97 (m, 3H), 6.74-6.66 (m, 1H), 6.47 (t,J_(H-F)=74.03 Hz, 1H), 5.45 (brs, 2H), 3.11 (s, 3H), 2.25 (s, 3H); LC(260 nm): R=3.919 min, LC Purity: 96.1%, m/z (M+1): 382, LC (220 nm):R=3.922 min, LC Purity: 96.8%.

Example 4 Synthesis of FAH-5:2-amino-4-(3,5-difluorophenyl)-4-(3,5-dimethylphenyl)-1-methyl-1H-imidazol-5(4H)-oneSynthesis of 2-(3,5-difluorophenyl)-1,3-dithiane

BF₃.OMe₂ (2.50 mL, 27.5 mmol) was added dropwise to a solution of1,3-propanedithiol (3.70 mL, 36.6 mmol) and 3,5-difluorobenzaldehyde(4.10 mL, 36.6 mmol) in DCM (75 mL) at 0° C. The reaction was stirred atambient temperature for 1 hour where TLC (5% EtOAc/hexane) indicated acomplete reaction. The reaction mixture was then diluted with additionalDCM (50 mL), filtered through Celite, and the Celite pad was washed withadditional DCM (3×50 mL). The filtrate was washed with saturated NaHCO₃(3×100 mL), 10% KOH solution (2×100 mL), water (100 mL) and brine (100mL) and finally dried over sodium sulfate. The organic extract wasfiltered through a pad of silica and the silica pad washed with 10%Ethyl acetate/hexane mixtures (3×20 mL). The organic extract wasevaporated to afford 2-(3,5-difluorophenyl)-1,3-dithiane (8.44 g, 36.3mmol, 99%) as a crystalline white solid. The NMR was consistent with theproposed structure.

Synthesis of(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)(3,5-dimethylphenyl)methanol

2-(3,5-Difluorophenyl)-1,3-dithiane (8.00 g, 34.4 mmol) was dissolved in100 mL of dry THF and cooled to −10° C. BuLi (1.6M, 34 mL, 54.4 mmol)was added dropwise under nitrogen and the mixture was stirred for 15 minat −10° C. to afford a brown solution. A solution of3,5-dimethylbenzaldehyde (4.84 g, 36.1 mmol) in THF (10 mL) was addeddropwise and the reaction mixture was stirred for 15 minutes, thenwarmed to ambient temperature over 30 minutes and quenched withsaturated ammonium chloride solution. The organic phase was washed withbrine and dried with sodium sulfate. The solvent was removed and theresidue purified by flash chromatography (10% EtOAc/hexane) to give(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)(3,5-dimethylphenyl)methanol(6.60 g, 18.01 mmol, 52%) as a thick oil that solidified on standing.The NMR was consistent with the proposed structure.

Synthesis of1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)-2-hydroxyethanone

(2-(3,5-Difluorophenyl)-1,3-dithian-2-yl)(3,5-dimethylphenyl)methanol(6.60 g, 18.01 mmol) was dissolved in a solution of acetonitrile (75 mL)and water (15 mL). Bis(trifluoroacetoxy)iodobenzene (9.68 g, 22.51 mmol)was added in several portions to the vigorously stirred solution atambient temperature. After 60 minutes, TLC (20% EtOAC/hexane) analysisappeared to indicate a complete reaction. Ethyl acetate (150 mL) wasadded and the mixture was rinsed with saturated sodium bicarbonatesolution (2×50 mL) and brine (50 mL). The organic fractions were driedover sodium sulfate, filtered and evaporated. The residue was purifiedtwice by flash column chromatography (10% EtOAc/hexane) to give1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)-2-hydroxyethanone (2.10 g,7.60 mmol, 42%, ca. 90% purity by NMR) as a pale yellow solidcontaminated with starting material (ca. 10%); R_(f) (10% EtOAc/hexane):0.20 was identical for both starting material and product. However theNMR was consistent with the proposed structure of the product which wasthe major component.

Synthesis of1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)ethane-1,2-dione

1-(3,5-Difluorophenyl)-2-(3,5-dimethylphenyl)-2-hydroxyethanone (2.10 g,7.60 mmol) was dissolved in 80% acetic acid (10 mL) together withdiacetoxycopper hydrate (0.15 g, 0.76 mmol) and ammonium nitrate (0.46g, 5.70 mmol). The mixture was refluxed for 90 minutes and then cooled.The green coloured reaction mixture was poured into ethyl acetate (50mL) and washed with brine (2×25 mL), dried over sodium sulfate, filteredand evaporated. The residue was subjected to flash chromatography (20%EtOAc/hexane) to afford1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)ethane-1,2-dione (1.13 g,4.12 mmol, 54%) as a yellow solid. The NMR was consistent with theproposed structure.

Synthesis of2-amino-4-(3,5-difluorophenyl)-4-(3,5-dimethylphenyl)-1-methyl-1H-imidazol-5(4H)-one(FAH5)

A mixture of1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)ethane-1,2-dione (500 mg,1.82 mmol) in ethanol (15 mL) and dioxane (15 mL) was added1-methylguanidine hydrochloride (799 mg, 7.29 mmol) and stirred atambient temperature for 15 minutes. Sodium carbonate (773 mg, 7.29 mmol)in water (5 mL) was added and the mixture immersed into an oil bath at85° C. and stirred for 4 hours. TLC (EtOAc) indicated a completereaction. The reaction mixture was cooled to ambient temperature andconcentrated. The residue was purified twice by column chromatography(EtOAc, 50% EtOAc/hexane) and finally by PTLC (Chloroform) to afford2-amino-4-(3,5-difluorophenyl)-4-(3,5-dimethylphenyl)-1-methyl-1H-imidazol-5(4H)-one(0.20 g, 0.61 mmol, 33%) as a white solid after drying under high vacuumat 60° C. for 36 hours. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.07 (d, J 6.83Hz, 2H), 7.02 (brs, 2H), 6.91 (brs, 1H), 6.69 (t, J 8.48 Hz, 1H), 5.22(s, 2H), 3.10 (s, 3H), 2.27 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ ppm164.1, 163.9, 161.6, 161.5, 155.3, 145.4, 140.4, 138.2, 129.7, 124.5,110.5, 110.4, 110.3, 110.2, 103.0, 77.2, 25.9, 21.41, (please note: dueto presence of fluorine atoms, J² _(C-F)-J⁴ _(C-F) couplings giving riseto poorly resolved triplets and doublets are noted); LC (230 nm) R_(t)(min)=3.97, LC purity=95.29%; m/z: found [M+H]⁺=330.1, expected[M+H]⁺=330.1 (C₁₈H₁₈F₂N₃O).

Example 5 Synthesis of FAH-4 (ITH002329) Synthesis of2-(3,5-difluorophenyl)-1,3-dithiane

BF3.OMe2 (1.40 ml, 15.25 mmol) was added dropwise to a solution of1,3-propanedithiol (1.81 ml, 17.87 mmol) and 3,5-difluorobenzaldehyde(2.00 ml, 17.87 mmol) in DCM (50 ml) at 0° C. The reaction was stirredat ambient temperature for 1 hour where TLC (5% EtOAc/hexane) indicateda complete reaction. The reaction mixture was then diluted withadditional DCM (50 ml), filtered through Celite, and the Celite pad waswashed with additional DCM (3×20 ml). The filtrate was washed withsaturated NaHCO3 (3×50 ml), 10% KOH solution (2×50 ml), water (2×50 ml)and brine (50 ml) and finally dried over sodium sulfate. The organicextract was filtered and evaporated to afford2-(3,5-difluorophenyl)-1,3-dithiane (4.12 g, 17.73 mmol, 99%) as a whitesolid.

Synthesis of 3-(difluoromethoxy)benzaldehyde

A solution of sodium chlorodifluoroacetate (12.48 g, 82 mmol) and3-hydroxybenzaldehyde (5.00 g, 40.9 mmol) in DMF (75 ml) was added over3 hours to a solution of DMF (25 ml) containing potassium carbonate(8.49 g, 61.4 mmol) at 95° C. The reaction was allowed to age for anadditional 2 hours and then cooled. The reaction mixture was dilutedwith water (100 ml) and extracted with ethyl acetate (4×50 ml). Theorganic extract was washed with 10% (m/v) aqueous LiCl solution (3×25ml), dried over sodium sulfate, filtered and evaporated to give aresidue that was flash chromatographed (15% EtOAc/hexane) to give3-(difluoromethoxy)benzaldehyde (2.50 g, 14.52 mmol, 36%) as a yellowoil. The NMR was consistent with the proposed structure.

Synthesis of(3-(difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol

The 2-(3,5-difluorophenyl)-1,3-dithiane (3.37 g, 14.52 mmol) wasdissolved in 30 ml of dry THF and cooled to −10° C. BuLi (1.6M, 12.0 ml,19.20 mmol) was added dropwise under nitrogen and the mixture wasstirred for 15 min at −10° C. to afford a blood-red solution. A solutionof 3-(difluoromethoxy)benzaldehyde (2.50 g, 14.52 mmol) in THF (10 ml)was added dropwise and the mixture was stirred for 15 minutes, thenwarmed to ambient temperature over 10 minutes and quenched withsaturated ammonium chloride solution. The organic phase was washed withbrine and dried with sodium sulfate. The solvent was removed and theresidue flash chromatographed (10% EtOAc/hexane) to give(3-(difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol(3.33 g, 8.23 mmol, 57%) as a thick oil that solidified on standing. TheNMR was consistent with the proposed structure.

Synthesis of2-(3-(difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone

(3-(Difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol(3.33 g, 8.23 mmol) was dissolved in 15 ml of acetonitrile and 2.5 ml ofwater. Bis(trifluoroacetoxy)iodobenzene (4.43 g, 10.29 mmol) in 10 ml ofacetonitrile was slowly added to the vigorously stirred solution atambient temperature. After 30 minutes, TLC (20% EtOAC/hexane) analysisindicated a complete reaction. EtOAc (150 ml) was added and the mixturewas rinsed with saturated sodium bicarbonate solution (50 ml) and brine(50 ml). The organic fractions were dried, and the solvent was removedin vacuo. The crude product was purified twice by flash columnchromatography (10% EtOAc/hexane) to give2-(3-(difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone(1.01 g, 3.21 mmol, 39%) as a pale yellow oil. The NMR was consistentwith the proposed structure.

Synthesis of1-(3-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione

2-(3-(Difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone(1.00 g, 3.18 mmol) was dissolved in 80% acetic acid (10 ml) togetherwith diacetoxycopper hydrate (0.13 g, 0.64 mmol) and ammonium nitrate(0.19 g, 2.39 mmol). The mixture was refluxed for 90 minutes and thencooled. The copper coloured reaction mixture was poured into ethylacetate (50 ml) and washed with brine (2×25 ml), dried over sodiumsulfate, filtered and evaporated. The residue was subjected to flashchromatography (20% EtOAc/hexane) to afford1-(3-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione(0.46 g, 1.48 mmol, 47%) as a yellow oil. Further elution of the columnafforded starting material (0.40 g, 1.27 mmol, 40% recovery) as an oil.The desired product was used as received.

Synthesis of2-amino-4-(3-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one

A mixture of1-(3-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione(463 mg, 1.48 mmol) in ethanol (15 ml) and dioxane (15 ml) was added1-methylguanidine hydrochloride (650 mg, 5.93 mmol) and stirred atambient temperature for 15 minutes. Sodium carbonate (629 mg, 5.93 mmol)in water (5 ml) was added and the mixture immersed into an oil bath at85° C. and stirred for 3 hours. TLC (EtOAc) indicated a completereaction. The reaction mixture was cooled to ambient temperature andconcentrated. The residue was purified twice by PTLC (EtOAc) to afford2-amino-4-(3-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one(0.22 g, 0.60 mmol, 40%) as a yellow solid after drying under highvacuum at 60° C. for 48 hours.

¹H NMR (400 MHz, CDCl₃) δ ppm 7.38-7.29 (m, 2H), 7.24 (broad m, 1H),7.10-7.00 (m, 3H), 6.71 (m, 1H), 6.49 (t, J_(H)-F=74.03 Hz, 1H), 5.61(brs, 2H), 3.10 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ ppm 178.30, 164.13,164.00, 161.66, 161.53, 156.03, 151.27, 151.24, 151.21, 145.00, 144.92,144.83, 142.88, 129.90, 123.81, 118.73, 118.42, 118.17, 115.84, 113.25,110.30, 110.22, 110.11, 110.03, 103.47, 103.22, 102.97, 74.92, 25.95(please note: due to presence of fluorine atoms, J² _(C-F)-J⁴ _(C-F)couplings giving rise to triplets and doublets are noted); LC (220 nm):R_(t)=3.85 min, LC Purity: 95.6%, m/z [M]⁺=367.9,

Example 6 FAH-17:2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-fluorophenyl)-1-methyl-1H-imidazol-5(4H)-oneStep 1: Synthesis of 2-(4-(difluoromethoxy)-3-methylphenyl)-1,3-dithiane

BF₃.OEt₂ (4.30 ml, 34.8 mmol) was added dropwise to a solution of1,3-propanedithiol (4.07 ml, 40.3 mmol) and 3-fluorobenzaldehyde (5.00g, 40.3 mmol) in DCM (201 ml) at 0° C. The reaction was stirred atambient temperature for 1 hour where TLC (5% EtOAc/hexane) indicated acomplete reaction. The reaction mixture was then diluted with DCM (50ml), filtered through Celite (and the Celite pad was washed withadditional DCM (3×50 ml)) and the filtrate washed with brine (100 ml),saturated NaHCO3 (3×100 ml), 10% KOH solution (100 ml), water (100 ml)and brine (100 ml) and finally dried over sodium sulfate. The organicextract was filtered and evaporated. The product was purified using 5%ethyl acetate:hexane to afford 2-(3-fluorophenyl)-1,3-dithiane (8.71 g,39.0 mmol, 97%) as an off-clear oil. The oil was used directly into thenext step. The NMR was consistent with the proposed structure.

Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 ml) wasadded over 3 hours to a solution of DMF (15 ml) containing potassiumcarbonate (1.77 g, 12.78 mmol) at 95° C. The reaction was allowed to agefor an additional 15 minutes and then cooled. The reaction mixture wasdiluted with water (50 ml) and extracted with ethyl acetate (3×50 ml).The organic extract was washed with 10% (m/v) aqueous LiCl solution(3×25 ml), dried over sodium sulfate, filtered and evaporated to give aresidue that was flash chromatographed (15% EtOAc/hexane) to give4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37mmol, 63%) as a yellow oil. This oil was combined with that of theprevious experiment and passed through a Pasteur pipette column elutingwith 10% EtOAC/hexane to give an oil that solidified on standing (1.315g, 7.06 mmol, 67% over the two reactions). The proton NMR was consistentwith the proposed structure.

Finally, repeating the experiment using the conditions in attempt 2, anadditional 1.4 g of the desired product was isolated.

Step 3: Synthesis of(2-(4-(difluoromethoxy)-3-methylphenyl)-1,3-dithian-2-yl)(3-fluorophenyl)methanol

2-(3-fluorophenyl)-1,3-dithiane (4.00 g, 18.66 mmol) was dissolved indry THF (93.5 mL) and cooled to −10° C. nBuLi (1.6M, 14.00 ml, 22.40mmol) was added dropwise under nitrogen and the mixture was stirred for30 min at −10° C. to afford a dark red solution. A solution of4-(difluoromethoxy)-3-methylbenzaldehyde (3.47 g, 18.66 mmol) in THF(93.5 ml) was added dropwise and the mixture at −10° C. and was stirredfor 15 minutes, then warmed to ambient temperature over 1 h and quenchedwith saturated ammonium chloride solution (7.5 ml) followed by dilutionwith EtOAc (50 ml). The organic phase was washed with water (2×20 ml),brine (1×20 ml) and dried with sodium sulfate. After filtration andconcentration the crude product was purified by flash columnchromatography (15% EtOAc/Hex) to give(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluorophenyl)-1,3-dithian-2-yl)methanol(6.00 g, 14.96 mmol, 80%) as an oil.

Step 4: Synthesis of2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluorophenyl)-2-hydroxyethanone

(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-difluorophenyl)-1,3-dithian-2-yl)methanol(3.07 g, 7.34 mmol) was dissolved in acetonitrile (15 ml) and water (2.5ml). Bis(trifluoroacetoxy)iodobenzene (3.94 g, 9.17 mmol) inacetonitrile (10 ml) was slowly added at ambient temperature to thevigorously stirred solution. After 20 minutes TLC (20% EtOAc/hexane)analysis indicated a complete reaction. EtOAc (150 ml) was added and themixture was rinsed with saturated sodium bicarbonate solution (50 ml)and brine (50 ml). The organic fractions were dried, and the solvent wasremoved in vacuo. The crude product was purified twice by flash columnchromatography (10% EtOAc/hexane) to give2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluorophenyl)-2-hydroxyethanone(0.853 g, 2.60 mmol, 35%) as a pale yellow oil. The proton NMR wasconsistent with the proposed structure.

Step 5: Synthesis of1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione

1-(2,4-difluorophenyl)-2-(4-methoxy-3-fluorophenyl)ethane-1,2-dione wassynthesized according to the representative procedure using1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)-2-hydroxyethanone(0.500 g, 1.612 mmol) and gave1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione(0.3881 g, 78%) as a yellow solid. The NMR was consistent with theproposed structure.

Step 6: Synthesis of2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-fluorophenyl)-1-methyl-1H-imidazol-5(4H)-one

Potassium carbonate (0.516 g, 4.87 mmol) in water (4.6 mL) was addedinto a mixture of1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione(0.375 g, 1.217 mmol), 1-methylguanidine hydrochloride (0.533 g, 4.87mmol), dioxane (19 mL), and ethyl alcohol (25 mL). The reaction mixturewas stirred at 85° C. for 4 h. The volatiles were removed in vacuo, andthe residue was taken in chloroform (100 ml) and washed with water (2×25mL). The organic extracts were dried over MgSO₄. Evaporation andpurification by flash chromatography (60% EtOAc/Hex to 100% EtOAc)followed by re-crystallization from CHCl₃/hexanes gave2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluorophenyl)-1-methyl-1H-imidazol-5(4H)-one(216 mg, 47%) as a off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.37-7.13(m, 5H), 6.96 (m, 2H), 6.46 (t, J=74.1 Hz, 1H), 5.73 (s, 2H), 3.09 (s,3H), 2.23 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 178.87, 163.90, 161.46,155.70, 149.21, 143.36, 138.01, 130.04, 130.02-129.79 (m), 125.73,122.70 (d, J⁼2.9 Hz), 118.75, 116.17, 114.45 (dd, J⁼38.7, 22.0 Hz),113.60, 75.07, 25.87, 16.35. (please note: due to presence of fluorineatoms, J² _(C-F)-J⁴ _(C-F) couplings giving rise to poorly resolvedtriplets and doublets are noted); LC (260 nm) R_(t) (min)=3.923, LCpurity=96%; m/z: found [M+H]⁺=364.2, expected [M+H]⁺=364.3(C₁₇H₁₄F₃N₃O).

Example 7 FAH-17 HCl Salt

2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluorophenyl)-1-methyl-1H-imidazol-5(4H)-one(0.50 g, 1.38 mmol) was dissolved in anhydrous DCM (66 ml) followed byaddition of HCl (1M in diethyl ether, 2.2 ml). The mixture was stirredat room temperature for 5 min and the solvent evaporated in vacuo toyield2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluorophenyl)-1-methyl-1H-imidazol-5(4H)-one hydrochloride (0.50 g, 1.20 mmol, 87%) as a white solid. ¹H NMR(d₆-DMSO): 11.78 (brs, 1H), 9.73 (brs, 1H), 7.53-7.06 (m, 8H), 3.19 (s,3H), 2.23 (s, 3H); ¹³C NMR (d₆-DMSO): 176.62, 176.99, 172.57, 163.63,161.20, 157.95, 150.07, 150.04, 140.36, 140.30, 134.40, 129.86, 126.60,123.70, 119.52, 119.00, 116.96, 116.47, 116.26, 114.64, 114.41, 70.30,27.43, 16.40 (please note: due to presence of fluorine atoms, J²_(C-F)-J⁴ _(C-F) couplings giving rise to poorly resolved triplets anddoublets are noted); LC (220 nm): R_(t)=3.84 min, purity 96.5%; MS: ForC₁₈H₁₆F₃N₃O₂ expect [M+H]⁺=364.3 obtained 364.1

Example 8 Synthesis of FAH-22 Synthesis of2-(3-fluoro-5-methylphenyl)-1,3-dithiane

BF₃.OEt₂ (2.61 ml, 21.16 mmol) was added dropwise to a solution of1,3-propanedithiol (2.48 ml, 24.47 mmol) and3-fluoro-5-methylbenzaldehyde (3.38 g, 24.47 mmol) in DCM (122 ml) at 0°C. The reaction was stirred at ambient temperature for 1 hour where TLC(5% EtOAc/hexane) indicated a complete reaction. The reaction mixturewas then diluted with DCM (100 ml), filtered through Celite (and theCelite pad was washed with additional DCM (3×100 ml)) and the filtratewashed with brine (100 ml), saturated NaHCO3 (3×100 ml), 10% KOHsolution (100 ml), water (100 ml) and brine (100 ml) and finally driedover sodium sulfate. The organic extract was filtered and evaporated toafford 2-(3-fluoro-5-methylphenly)-1,3-dithiane (4.69 g, 77%) as a lightpink solid. The product was used in the next step without furtherpurification. The NMR was consistent with the proposed structure.

Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 ml) wasadded over 3 hours to a solution of DMF (15 ml) containing potassiumcarbonate (1.77 g, 12.78 mmol) at 95° C. The reaction was allowed to agefor an additional 15 minutes and then cooled. The reaction mixture wasdiluted with water (50 ml) and extracted with ethyl acetate (3×50 ml).The organic extract was washed with 10% (m/v) aqueous LiCl solution(3×25 ml), dried over sodium sulfate, filtered and evaporated to give aresidue that was flash chromatographed (15% EtOAc/hexane) to give4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37mmol, 63%) as a yellow oil. This oil was combined with that of theprevious experiment and passed through a Pasteur pipette column elutingwith 10% EtOAC/hexane to give an oil that solidified on standing (1.315g, 7.06 mmol, 67% over the two reactions). The proton NMR was consistentwith the proposed structure. 1.4 g of the desired product was isolated.

Synthesis of(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dithian-2-yl)methanol

(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dithian-2-yl)methanolwas prepared according to the representative procedure using2-(3-fluoro-5-methylphenyl)-1,3-dithiane (0.932 g, 4.08 mmol) and4-(difluoromethoxy)-3-methylbenzaldehyde (0.760 g, 4.08 mmol) which gave(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dithian-2-yl)methanol(0.413 g, 24%) as a yellow oil. The NMR was consistent with the proposedstructure.

Synthesis of2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)-2-hydroxyethanone

2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)-2-hydroxyethanonewas synthesized according to the representative procedure using(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dithian-2-yl)methanol(0.400 g, 0.965 mmol) and gave2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)-2-hydroxyethanone(162 mg, 47%) as a yellow solid. The NMR was consistent with theproposed structure. Note:(4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dithian-2-yl)methanone(104 mg, 22%) was also recovered.

Synthesis of1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane-1,2-dione

1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane-1,2-dionewas synthesized according to the representative procedure using2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)-2-hydroxyethanone(0.150 g, 0.463 mmol) and gave1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane-1,2-dione(0.1134 g, 74%) as a yellow solid. The NMR was consistent with theproposed structure.

Synthesis of 2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluoro-5-methylphenyl)-1-methyl-1H-imidazol-5(4H)-one

Potassium carbonate (0.149 g, 1.407 mmol) in water (2.3 mL) was addedinto a mixture of1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane-1,2-dione(0.1134 g, 0.352 mmol), 1-methylguanidine hydrochloride (0.154 g, 1.407mmol), dioxane (5.46 mL), and ethyl alcohol (7.10 mL). The reactionmixture was stirred at 85° C. for 1.5 h. The volatiles were removed invacuo, and the residue was taken in chloroform (50 ml) and washed withwater (2×15 mL). The organic extracts were dried over MgSO₄. Evaporationand purification five times by flash chromatography (1% methanol inethyl acetate) gave2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluoro-5-methylphenyl)-1-methyl-1H-imidazol-5(4H)-one(85 mg, 75%) as a off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.30 (d,J=2.0 Hz, 1H), 7.28-7.20 (m, 1H), 7.02 (s, 1H), 6.95 (m, 2H), 6.77 (d,J=9.3 Hz, 1H), 6.45 (t, J=74.1 Hz, 1H), 5.26 (s, 2H), 3.07 (s, 3H), 2.28(s, 3H), 2.23 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 178.52, 163.88,161.44, 155.75, 149.22 (t, J=2.5 Hz), 141.88 (dd, J=288.6, 7.8 Hz),138.02, 130.05 (d, J=9.4 Hz), 125.75, 123.30 (d, J=2.5 Hz), 118.75 (d,J=7.5 Hz), 116.21, 115.32 (d, J=21.0 Hz), 113.63, 111.33 (d, J=23.3 Hz),74.72, 25.83, 21.46 (d, J=1.8 Hz), 16.33. (please note: due to presenceof fluorine atoms, J² _(C-F)-J⁴ _(C-F) couplings giving rise to poorlyresolved triplets and doublets are noted); LC (220 nm) R_(t)(min)=4.007, LC purity=98%; m/z: found [M+H]⁺=378.2, expected[M+H]⁺=378.4 (C₁₉H₁₈F₃N₃O₂).

Example 9 Synthesis of FAH-23:2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-chlorophenyl)-1-methyl-1H-imidazol-5(4H)-oneStep 1: Synthesis of 2-(3-chlorophenyl)-1,3-dithiane

BF₃.OEt₂ (2.28 ml, 18.46 mmol) was added dropwise to a solution of1,3-propanedithiol (2.16 ml, 21.34 mmol) and 3-chlorobenzaldehyde (3.00g, 21.34 mmol) in DCM (107 ml) at 0° C. The reaction was stirred atambient temperature for 1 hour where TLC (5% EtOAc/hexane) indicated acomplete reaction. The reaction mixture was then diluted with DCM (50ml), filtered through Celite (and the Celite pad was washed withadditional DCM (3×10 ml)) and the filtrate washed with brine (100 ml),saturated NaHCO3 (3×100 ml), 10% KOH solution (100 ml), water (100 ml)and brine (100 ml) and finally dried over sodium sulfate. The organicextract was filtered and evaporated to afford2-(3-chlorophenyl)-1,3-dithiane (4.62 g, 92%) as a colourless solid. Theproduct was used in the next step without further purification.

Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 ml) wasadded over 3 hours to a solution of DMF (15 ml) containing potassiumcarbonate (1.77 g, 12.78 mmol) at 95° C. The reaction was allowed to agefor an additional 15 minutes and then cooled. The reaction mixture wasdiluted with water (50 ml) and extracted with ethyl acetate (3×50 ml).The organic extract was washed with 10% (m/v) aqueous LiCl solution(3×25 ml), dried over sodium sulfate, filtered and evaporated to give aresidue that was flash chromatographed (15% EtOAc/hexane) to give4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37mmol, 63%) as a yellow oil. This oil was combined with that of theprevious experiment and passed through a Pasteur pipette column elutingwith 10% EtOAC/hexane to give an oil that solidified on standing (1.315g, 7.06 mmol, 67% over the two reactions). The proton NMR was consistentwith the proposed structure. 1.4 g of the desired product was isolated.

Step 3: Synthesis of(4-(difluoromethoxy)-3-methylphenyl)(2-(3-chlorophenyl)-1,3-dithian-2-yl)methanol

2-(3-chlorophenyl)-1,3-dithiane (0.92 g, 3.98 mmol) was dissolved in dryTHF (20 mL) and cooled to −29° C. nBuLi (1.6M, 2.99 ml, 4.78 mmol) wasadded dropwise under nitrogen and the mixture was stirred for 30 min at−29° C. to afford a dark red solution. A solution of4-(difluoromethoxy)-3-methylbenzaldehyde (0.74 g, 3.98 mmol) in THF(19.8 ml) was added dropwise and the mixture at −29° C. and was stirredfor 15 minutes, then warmed to ambient temperature over 1 h and quenchedwith saturated ammonium chloride solution (7.5 ml) followed by dilutionwith EtOAc (50 ml). The organic phase was washed with water (2×20 ml),brine (1×20 ml) and dried with sodium sulfate. After filtration andconcentration the crude product was purified by flash columnchromatography (15% EtOAc/Hexane) to give(2-(3-chlorophenyl)-1,3-dithian-2-yl)(4-(difluoromethoxy)-3-methylphenyl)methanol(0.81 g, 1.94 mmol, 49%) as an oil. The NMR was consistent with theproposed structure.

Step 4: Synthesis of2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-chlorophenyl)-2-hydroxyethanone

(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-chlorophenyl)-1,3-dithian-2-yl)methanol(3.07 g, 7.34 mmol) was dissolved in acetonitrile (15 ml) and water (2.5ml). Bis(trifluoroacetoxy)iodobenzene (3.94 g, 9.17 mmol) inacetonitrile (10 ml) was slowly added at ambient temperature to thevigorously stirred solution. After 20 minutes TLC (20% EtOAc/hexane)analysis indicated a complete reaction. EtOAc (150 ml) was added and themixture was rinsed with saturated sodium bicarbonate solution (50 ml)and brine (50 ml). The organic fractions were dried, and the solvent wasremoved in vacuo. The crude product was purified twice by flash columnchromatography (10% EtOAc/hexane) to give2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-chlorophenyl)-2-hydroxyethanone(0.803 g, 2.4 mmol, 32%) as a pale yellow oil. The proton NMR wasconsistent with the proposed structure.

Step 5: Synthesis of1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-chlorophenyl)ethane-1,2-dione

(2-(3-chlorophenyl)-1,3-dithian-2-yl)(4-(difluoromethoxy)-3-methylphenyl)methanol(0.80 g, 1.92 mmol) was dissolved in dichloromethane (24.29 ml) andtert-butanol (5.14 ml, 53.7 mmol) under nitrogen atmosphere. Dess-MartinPeriodinane (2.04 g, 4.80 mmol) was added and the reaction was stirredovernight at room temperature. Sodium thiosulphate (5 ml, 1M) was addedand the layers were separated. The organic phase was washed with sodiumhydrogen carbonate and the solvent was evaporated. Purification on prepplate in 25% ethyl acetate hexane gave1-(3-chlorophenyl)-2-(4-(difluoromethoxy)-3-methylphenyl)ethane-1,2-dione(0.41 g, 1.27 mmol, 66%) as a yellow solid was used directly into thenext stage.

Step 6: Synthesis of2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-chlorophenyl)-1-methyl-1H-imidazol-5(4H)-one

Potassium carbonate (0.522 g, 4.93 mmol) in water (7.85 mL) was addedinto a mixture of1-(3-chlorophenyl)-2-(4-(difluoromethoxy)-3-methylphenyl)ethane-1,2-dione(0.40 g, 1.23 mmol), 1-methylguanidine hydrochloride (0.54 g, 4.93mmol), dioxane (19.13 mL), and ethyl alcohol (24.87 mL). The reactionmixture was stirred at 85° C. for 1.5 h. The volatiles were removed invacuo, and the residue was taken in chloroform (50 ml) and washed withwater (2×15 mL). The organic extracts were dried over MgSO₄. Evaporationand purified three times on PTLC (1% MeOH in EtOAc) and columnchromatography (50-90% ethyl acetate: hexane) to give2-amino-4-(3-chlorophenyl)-4-(4-(difluoromethoxy)-3-methylphenyl)-1-methyl-1H-imidazol-5(4H)-one(0.20 g, 0.52 mmol, 43%) as an off-white solid. ¹H NMR (CDCl₃): 7.48 (s,1H), 7.30-7.20 (m, 5H), 7.0 (s, 1H), 6.48 (t, 1H, J=74.1 Hz), 4.50 (brs,2H), 3.12 (s, 3H), 2.26 (s, 3H); ¹³C NMR (CDCl₃): 178.45, 155.74,149.25, 143.22, 137.90, 134.32, 130.05, 127.12, 125.74, 125.36, 118.78,116.17, 113.60 74.73, 25.89, 16.35 (please note: due to presence offluorine atoms, J² _(C-F)-J⁴ _(C-F) couplings giving rise to poorlyresolved triplets and doublets are noted); LC (220 nm): R_(t)=3.95 min,purity 96.6%; MS: For C₁₈H₁₆ClF₂N₃O₂ expect [M+H]⁺=380.8 obtained 380.1

Example 10 Synthesis of Compound FAH-27:2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-methylphenyl)-1-methyl-1H-imidazol-5(4H)-oneStep 1: Synthesis of 2-(3-methylphenyl)-β-dithiane

BF₃.OEt₂ (2.67 ml, 21.60 mmol) was added dropwise to a solution of1,3-propanedithiol (2.53 ml, 24.97 mmol) and 3-methylbenzaldehyde (3.00g, 24.97 mmol) in DCM (125 ml) at 0° C. The reaction was stirred atambient temperature for 1 hour where TLC (5% EtOAc/hexane) indicated acomplete reaction. The reaction mixture was then diluted with DCM (50ml), filtered through Celite (and the Celite pad was washed withadditional DCM (3×10 ml)) and the filtrate washed with brine (100 ml),saturated NaHCO3 (3×100 ml), 10% KOH solution (100 ml), water (100 ml)and brine (100 ml) and finally dried over sodium sulfate. The organicextract was filtered and evaporated to afford 2-(m-tolyl)-1,3-dithiane(4.66 g, 85%) as a light brown solid. The product was used in the nextstep without further purification. The proton NMR was consistent withthe proposed structure.

Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 ml) wasadded over 3 hours to a solution of DMF (15 ml) containing potassiumcarbonate (1.77 g, 12.78 mmol) at 95° C. The reaction was allowed to agefor an additional 15 minutes and then cooled. The reaction mixture wasdiluted with water (50 ml) and extracted with ethyl acetate (3×50 ml).The organic extract was washed with 10% (m/v) aqueous LiCl solution(3×25 ml), dried over sodium sulfate, filtered and evaporated to give aresidue that was flash chromatographed (15% EtOAc/hexane) to give4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37mmol, 63%) as a yellow oil. This oil was combined with that of theprevious experiment and passed through a Pasteur pipette column elutingwith 10% EtOAC/hexane to give an oil that solidified on standing (1.315g, 7.06 mmol, 67% over the two reactions). The proton NMR was consistentwith the proposed structure. 1.4 g of the desired product was isolated.

Step 3: Synthesis of(4-(difluoromethoxy)-3-methylphenyl)(2-(3-methylphenyl)-1,3-dithian-2-yl)methanol

2-(m-Tolyl)-1,3-dithiane (2.00 g, 9.51 mmol) was dissolved in dry THF(47.5 mL) and cooled to −29° C. nBuLi (1.6M, 7.13 ml, 11.41 mmol) wasadded dropwise under nitrogen and the mixture was stirred for 30 min at−10° C. to afford a dark red solution. A solution of4-(difluoromethoxy)-3-methylbenzaldehyde (1.77 g, 9.51 mmol) in THF(47.5 ml) was added dropwise and the mixture at −29° C. and was stirredfor 15 minutes, then warmed to ambient temperature over 1 h and quenchedwith saturated ammonium chloride solution (7.5 ml) followed by dilutionwith EtOAc (50 ml). The organic phase was washed with water (2×20 ml),brine (1×20 ml) and dried with sodium sulfate. After filtration andconcentration the crude product was purified by flash columnchromatography (15% EtOAC/Hex) to give(4-(difluoromethoxy)-3-methylphenyl)(2-(m-tolyl)-1,3-dithian-2-yl)methanol(2.73 g, 6.88 mmol, 72%) as an oil. The NMR was consistent with theproposed structure.

Step 4: Synthesis of2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-methylphenyl)-2-hydroxyethanone

(4-(Difluoromethoxy)-3-methylphenyl)(2-(m-tolyl)-1,3-dithian-2-yl)methanol(2.70 g, 6.81 mmol) was dissolved in dichloromethane (86 ml) andtert-butanol (18.28 ml, 191 mmol) under nitrogen atmosphere. Dess-MartinPeriodinane (7.22 g, 17.02 mmol) was added and the reaction was stirredovernight at room temperature. Sodium thiosulphate (5 ml, 1M) was addedand the layers were separated. The organic phase was washed with sodiumhydrogen carbonate and the solvent was evaporated. Purification oncolumn chromatography in 5% ethyl acetate/hexane gave1-(4-(difluoromethoxy)-3-methylphenyl)-2-(m-tolyl)ethane-1,2-dione (1.44g, 4.75 mmol, 70%) as a yellow solid.

Step 5: Synthesis of1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-methylphenyl)ethane-1,2-dione

(4-(Difluoromethoxy)-3-methylphenyl)(2-(m-tolyl)-1,3-dithian-2-yl)methanol(2.70 g, 6.81 mmol) was dissolved in dichloromethane (86 ml) andtert-butanol (18.28 ml, 191 mmol) under nitrogen atmosphere. Dess-MartinPeriodinane (7.22 g, 17.02 mmol) was added and the reaction was stirredovernight at room temperature. Sodium thiosulphate (5 ml, 1M) was addedand the layers were separated. The organic phase was washed with sodiumhydrogen carbonate and the solvent was evaporated. Purification oncolumn chromatography in 5% ethyl acetate/hexane gave1-(4-(difluoromethoxy)-3-methylphenyl)-2-(m-tolyl)ethane-1,2-dione (1.44g, 4.75 mmol, 70%) as a yellow solid and was used directly into the nextstage.

Step 6: Synthesis of2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-methylphenyl)-1-methyl-1H-imidazol-5(4H)-one

Potassium carbonate (1.81 g, 17.09 mmol) in water (27.23 mL) was addedinto a mixture of1-(4-(difluoromethoxy)-3-methylphenyl)-2-(m-tolyl)ethane-1,2-dione (1.30g, 4.27 mmol), 1-methylguanidine hydrochloride (1.87 g, 17.09 mmol),dioxane (66.3 mL), and ethyl alcohol (86 mL). The reaction mixture wasstirred at 85° C. for 1.5 h. The volatiles were removed in vacuo, andthe residue was taken in chloroform (50 ml) and washed with water (2×15mL). The organic extracts were dried over MgSO₄. Evaporation andpurified three times by PTLC (1% MeOH in EtOAc) and once by columnchromatography (50-90% ethyl acetate: hexane) to give2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-1-methyl-4-(m-tolyl)-1H-imidazol-5(4H)-one(0.39 g, 1.07 mmol, 25%) as an off-white solid. ¹H NMR (CDCl₃): 7.34 (s,1H), 7.24-7.10 (m, 5H), 6.95 (d, 1H, J=8 Hz), 6.47 (t, 1H, J=74.1 Hz),6.05 (brs, 2H), 3.04 (s, 3H), 2.30 (s, 3H), 2.23 (s, 3H); ¹³C NMR(CDCl₃): 178.55, 155.97, 149.14, 149.12, 149.09, 141.16, 138.14, 130.20,128.35, 127.56, 125.94, 124.16, 118.83, 118.61, 116.25, 113.67, 74.74,25.70, 21.52, 16.32 (please note: due to presence of fluorine atoms, J²_(C-F)-J⁴ _(C-F) couplings giving rise to poorly resolved triplets anddoublets are noted); LC (220 nm): R_(t)=3.85 min, purity 97.3%; MS: ForC₁₉H₁₉F₂N₃O₂ expect [M+H]⁺=360.4 obtained 360.2

Example 11 Synthesis of FAH-28:2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-(trifluoromethyl)phenyl)-1-methyl-1H-imidazol-5(4H)-oneStep 1: Synthesis of 2-(3-trifluoromethyl)phenyl)-β-dithiane

BF₃.OEt₂ (1.84 ml, 14.90 mmol) was added dropwise to a solution of1,3-propanedithiol (1.74 ml, 17.23 mmol) and3-(trifluoromethyl)benzaldehyde (3.00 g, 17.23 mmol) in DCM (86 ml) at0° C. The reaction was stirred at ambient temperature for 1 hour whereTLC (5% EtOAc/hexane) indicated a complete reaction. The reactionmixture was then diluted with DCM (50 ml), filtered through Celite (andthe Celite pad was washed with additional DCM (3×10 ml)) and thefiltrate washed with brine (100 ml), saturated NaHCO3 (3×100 ml), 10%KOH solution (100 ml), water (100 ml) and brine (100 ml) and finallydried over sodium sulfate. The organic extract was filtered andevaporated to afford 2-(3-(trifluoromethyl)phenyl)-1,3-dithiane (4.62 g,17.30 mmol, 100%) as a colourless solid. The product was used in thenext step without further purification.

Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 ml) wasadded over 3 hours to a solution of DMF (15 ml) containing potassiumcarbonate (1.77 g, 12.78 mmol) at 95° C. The reaction was allowed to agefor an additional 15 minutes and then cooled. The reaction mixture wasdiluted with water (50 ml) and extracted with ethyl acetate (3×50 ml).The organic extract was washed with 10% (m/v) aqueous LiCl solution(3×25 ml), dried over sodium sulfate, filtered and evaporated to give aresidue that was flash chromatographed (15% EtOAc/hexane) to give4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37mmol, 63%) as a yellow oil. This oil was combined with that of theprevious experiment and passed through a Pasteur pipette column elutingwith 10% EtOAC/hexane to give an oil that solidified on standing (1.315g, 7.06 mmol, 67% over the two reactions). The proton NMR was consistentwith the proposed structure. 1.4 g of the desired product was isolated.

Step 3: Synthesis of(4-(difluoromethoxy)-3-methylphenyl)(2-(3-(trifluoromethyl)phenyl)-1,3-dithian-2-yl)methanol

2-(3-(trifluoromethyl)phenyl)-1,3-dithiane (2.00 g, 7.57 mmol)(021STM-080) was dissolved in dry THF (38 mL) and cooled to −29° C.nBuLi (1.6M, 5.67 ml, 9.08 mmol) was added dropwise under nitrogen andthe mixture was stirred for 30 min at −29° C. to afford a dark redsolution. A solution of 4-(difluoromethoxy)-3-methylbenzaldehyde (1.41g, 7.57 mmol) in THF (38 ml) was added dropwise and the mixture at −29°C. and was stirred for 15 minutes, then warmed to ambient temperatureover 1 h and quenched with saturated ammonium chloride solution (7.5 ml)followed by dilution with EtOAc (50 ml). The organic phase was washedwith water (2×20 ml), brine (1×20 ml) and dried with sodium sulfate.After filtration and concentration the crude product was purified byflash column chromatography (5-15% EtOAc/Hex) to give(4-(difluoromethoxy)-3-methylphenyl)(2-(3-(trifluoromethyl)phenyl)-1,3-dithian-2-yl)methanol(2.29 g, 4.55 mmol, 60%) as a golden oil.

Step 4: Synthesis of2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-(trifluoromethyl)phenyl)-2-hydroxyethanone

(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-trifluoromethylphenyl)-1,3-dithian-2-yl)methanol(3.07 g, 7.34 mmol) was dissolved in acetonitrile (15 ml) and water (2.5ml). Bis(trifluoroacetoxy)iodobenzene (3.94 g, 9.17 mmol) inacetonitrile (10 ml) was slowly added at ambient temperature to thevigorously stirred solution. After 20 minutes TLC (20% EtOAc/hexane)analysis indicated a complete reaction. EtOAc (150 ml) was added and themixture was rinsed with saturated sodium bicarbonate solution (50 ml)and brine (50 ml). The organic fractions were dried, and the solvent wasremoved in vacuo. The crude product was purified twice by flash columnchromatography (10% EtOAc/hexane) to give2-(4-(difluoromethoxy)-3-trifluoromethylphenyl)-1-(3-methylphenyl)-2-hydroxyethanone(0.803 g, 2.4 mmol, 32%) as a pale yellow oil. The proton NMR wasconsistent with the proposed structure.

Step 5: Synthesis of1-(4-(difluoromethoxy)-3-(trifluoromethyl)phenyl)-2-(3-(trifluoromethyl)phenyl)ethane-1,2-dione

(4-(difluoromethoxy)-3-methylphenyl)(2-(3-(trifluoromethyl)phenyl)-1,3-dithian-2-yl)methanol(2.20 g, 4.88 mmol) was dissolved in dichloromethane (61.8 ml) andtert-butanol (13.08 ml, 137 mmol) under nitrogen atmosphere. Dess-MartinPeriodinane (5.18 g, 12.21 mmol) was added and the reaction was stirredovernight at room temperature. Sodium thiosulphate (5 ml, 1M) was addedand the layers were separated. The organic phase was washed with sodiumhydrogen carbonate and the solvent was evaporated. Purification oncolumn chromatography in 5% ethyl acetate/hexane gave1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-(trifluoromethyl)phenyl)ethane-1,2-dione(1.20 g, 3.35 mmol, 69%) as a yellow solid was used directly into thenext stage.

Step 6: Synthesis of2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-(trifluoromethyl)phenyl)-1-methyl-1H-imidazol-5(4H)-one

Potassium carbonate (1.36 g, 12.84 mmol) in water (20.46 mL) was addedinto a mixture of1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-(trifluoromethyl)phenyl)ethane-1,2-dione(1.15 g, 3.21 mmol), 1-methylguanidine hydrochloride (1.41 g, 12.84mmol), dioxane (49.8 mL), and ethyl alcohol (64.8 mL). The reactionmixture was stirred at 85° C. for 1.5 h. The volatiles were removed invacuo, and the residue was taken in chloroform (50 ml) and washed withwater (2×15 mL). The organic extracts were dried over MgSO₄. Evaporationand purified three times by PTLC (1% MeOH in EtOAc) and once by columnchromatography (50-90% ethyl acetate: hexane) to give2-amino-4-(3,5-difluorophenyl)-4-(6-methoxypyridin-3-yl)-1-methyl-1H-imidazol-5(4H)-one(0.18 g, 0.44 mmol, 14%) as an off-white solid. ¹H NMR (CDCl₃): 7.80 (s,1H), 7.70 (d, 1H, J=8 Hz), 7.55 (d, 1H, J=8 Hz), 7.45-7.41 (m, 1H), 7.32(s, 1H), 7.28-7.24 (m, 1H), 7.0 (d, 1H, J=8 Hz), 6.48 (t, 1H, J=74.1Hz), 5.64 (brs, 2H), 3.10 (s, 3H), 2.26 (s, 3H); ¹³C NMR (CDCl₃):178.78, 156.18, 156.09, 156.07, 149.23, 142.39, 138.11, 130.71, 130.13,128.99, 125.72, 122.72, 118.73, 116.15, 113.57, 75.05, 25.84, 16.33(please note: due to presence of fluorine atoms, J² _(C-F)-J⁴ _(C-F)couplings giving rise to poorly resolved triplets and doublets arenoted); LC (220 nm): R_(t)=4.04 min, purity 96.6%; MS: For C₁₉H₁₆F₅N₃O₂expect [M+H]⁺=414.3 obtained 414.1

Example 12 SPR Analysis

The surfaces of all two flow cell FC1 and FC2 of acarboxymethylated-dextran (CM-5) chips were washed sequentially with 50mM NaOH, 1 mM HCl, 0.05% H₃PO₄ and 20 mM sodium phosphate pH 7.4, 125 mMsodium chloride in parallel using a flow rate of 30 l/min for 1 minusing a Biacore T-100 (GE Healthcare). The fusion protein wasimmobilized via amine coupling using 20 mM phosphate, 125 mM sodiumchloride pH 7.4 on to FC2. This fusion protein TRX-eAPP₅₇₅₋₆₂₄-containsa fusion of thioredoxin (TRX) and residues 575-624 of the APP ectodomain(20-kDa). The fusion protein is produced as described in Libeu et al(JAD 2011). The protein was concentrated to 2 mg/ml in 20 mM phosphatepH 6.5, 125 mM sodium chloride and then dissolved to a concentration of50 μg per ml in 20 mM sodium acetate pH 5.0. FC1 served as a referencecell following a mock immobilization with buffer alone. For all cells,the flow rate was 10 μl per min. The chip was blocked with 1Methanolamine (pH 8.5). The final RU values were determined for BACEinhibitors binding to TRX-eAPP₅₇₅₋₆₂₄ by flowing varying concentrationsof the inhibitor in DMSO to 50 μM. Compounds were diluted from 10 mMsolutions in DMSO to 50 μM in 1% DMSO, 20 mM sodium phosphate pH 7.4,125 mM sodium chloride, 0.05% Tween and then serially diluted by 1.5 for10 steps. Binding traces were recorded for each dilution with a bindingphase of 60 seconds and a dissociation phase of 240 seconds. Each cyclewas performed at 20° C. with a constant flow rate of 20 l/min. Anadditional 240 seconds of buffer flow at 60 μl per min across the cellswas applied as a regeneration phase to facilitate complete dissociationof the compound from the protein. The sensograms were obtained bysubtraction of the reference and buffer signals using the Biacore T100Evaluation software. The binding curves were modeled with the PRISM(Graphpad Inc).

Example 13 Experimental Methods for Measurement Aβ42 in SH-SY5Y Cells InVitro Abeta Testing Assay:

SH-SY5Y neuroblastoma cells were seeded at 50,000 cells/well in a 96wells plate for 24 h. Then their medium was changed for fresh mediumsupplemented with desired concentration of the hydantoin compound (e.g.compound 3). After 24 h, 20 μl of the medium was added to 2 μl of thecomplete protease inhibitor with 1 μM EDTA and kept at 4° C. untilanalysis using the ELISA assay below.

ELISA Assays:

ELISA kits from Invitrogen were also used to quantify Aβ31-42 (KHB3544)in duplicate. For the Aβ1-42 ultrasensitive ELISA, samples were diluted1:2 (50 μl CSF plus 50 μl kit-provided standard diluent buffer). Assayswere performed according to manufacturer's instructions. In short,standards and samples were added to a plate pre-coated with a monoclonalcapture antibody specific for the amino terminus of Hu Aβ. The sampleswere co-incubated with a rabbit detection antibody (Ab) specific for thecarboxy terminus of the Aβ species being assayed for 3 hr at roomtemperature overnight at 4°(Aβ1-42) with gentle rocking. After washing,bound rabbit Ab was detected using a horseradish peroxidase-labeledanti-rabbit secondary Ab. After washing again, bound HRP-anti rabbit Abwas detected colorimetrically (Spectramax 190, Molecular Devices) by theaddition of a substrate solution. 1 mM 4-(2-Aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF) protease inhibitor (101500, Calbiochem)was added to standards and samples.

Example 14 Brain Uptake Testing (PK)

In general, CNS exposure of the hydantoins were performed as follows:Studies consisted of collection of heparinized plasma and brains aftertreatment with the hydantoins, following subcutaneous (sc)administration of the molecules at 10 mg/kg. Plasma and brain levels ofthe compounds were determined by quantitative LC/MS/MS methodology,conducted at Integrated Analytical Solutions (on the internet atianalytical.net). Plasma samples were precipitated withacetonitrile:methanol (1:1) cocktail containing an internal standard.The brain samples were homogenized directly in ethylacetate or extractedfrom 5M guanidine homogenates using the liquid-liquid method. Theresulting supernatant were evaporated to dryness and subjected to theLC/MS/MS analysis. For each compound 3 mice were used for analysis. Thebrain-to-plasma ratios and brain levels were then be calculated.

Example 15 Selectivity of ABBI: Inhibition of APP-BACE Versus PSLG1-BACEor NRG1-BACE Cleavage

P5-P5′ Assay:

In order to determine the APP-BACE1 IC50 Sigma BACE1 substrate(7-Methoxycumarin-4-acetyl-[Asn670, Lue671]-Amyloid b/A4 PrecursorProtein 770 Fragment 667-676-(2,4 dinitrophenyl) Lys-Arg-Arg amidetrifluoroacetate salt) was used, manufacturer protocol was followed.Briefly, 0.01 units of BACE1 were incubated for 1 h at room temperaturewith a BACE inhibitor, then the substrate was added to each well and thefluorescence was read immediately and every 30 min for 2h. Activity wasdetermine by dividing the fluorescence at a specific [BACE inhibitor] bythe fluorescence at [BACE inhibitor]=0 μM, the % activity was plotted vslog[BACE inhibitor] to determine the APP-BACE1 using GraphPad Prism 5(FIG. 6A)

PSGL1 and NRG1 Assays:

Briefly, in order to determine the PSGL-1-BACE1 IC50 IC50 HEK 293 cellswere plated in 24 well plates and transiently cotransfected with eitherPSGL1/lacZ or NRG1/lacZ constructs using Lipofectamine 2000;manufacturer protocol was followed. Two hours after adding the DNA-lipidcomplex to the cells a BACE inhibitor was added to each well, then cellswere incubated overnight at 37° C. and 5% CO₂. Cultured medium wascollected to determine NRG1 or PSGL1, and cells were lysed to measurelacZ levels. Sigma SEAP kit standard protocol was conducted on thecultured medium to detect levels of PSGL1 or NRG1. Promega kitinstructions were followed to determine lacZ concentration. The ratio ofPSGL1/lacz vs [BACE inhibitor] or were plotted to determine theBACE1-IC50 on each of the substrate using GraphPad Prism 5 (FIG. 6B).The ABBI FAH17 shows a >200 fold selectivity for APP over PSGL1. Similartesting shows that FAH17 is >10 fold selective for APP over NRG1.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1: A compound of Formula I:

wherein: R⁷ is selected from the group consisting of C═O, C═S, C—NH₂,and C═NH, and the bond represented by the wavy line is a single bondwhen R⁷ is C═O, C═S, or C═NH, and a double bond when R⁷ is C—NH₂; R⁸ andR⁹ are independently selected from the group consisting of H, alkyl,cycloalkyl, and aryl, provided that when the bond represented by thewavy line is a double bond, then R⁹ is absent; R⁰ is selected from thegroup consisting of aryl, substituted aryl, disubstituted aryl,heteroaryl, substituted heteroaryl, disubstituted heteroaryl, alkyl,haloalkyl, cycloalkyl, alkenyl, and alkynyl; X¹ is selected from thegroup consisting of CH, and N; R⁵ and R⁶ are independently selected fromhalogen; R³ and R⁴ are independently absent or selected from the groupconsisting of alkyl, cycloalkyl, alkoxy, thioalkyl, and when X¹ is C,then R⁰ is not phenyl monosubstituted at the para position with —OCHF₂,or a pharmaceutically acceptable salt thereof, a tautomer thereof, apharmaceutically acceptable salt of a tautomer thereof, an enantiomerthereof, or a pharmaceutically acceptable salt of an enantiomer thereof.2: The compound of claim 1, wherein R⁷ is C═NH, or C═O.
 3. (canceled) 4:The compound of claim 2, wherein said compound is a compound of theformula:


5. (canceled) 6: The compound of claim 2, wherein R⁷ is C—NH₂ and saidcompound is a compound of Formula II:

7: The compound of claim 6, wherein said compound is a compound ofFormula III:

wherein: R¹ and R² are independently absent or selected from the groupconsisting of alkyl, haloalkyl, cycloalkyl, alkenyl, alkynyl, alkoxy,thioalkyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; and X², Y, and Z are independently CH or N. 8-10. (canceled)11: The compound of claim 7, wherein said compound is a compound ofFormula IV:

12-13. (canceled) 14: The compound of claim 1, wherein said compound isa compound selected from the group consisting of

15-17. (canceled) 18: A compound of Formula VI:

or a pharmaceutically acceptable salt thereof, a tautomer thereof, apharmaceutically acceptable salt of a tautomer thereof, an enantiomerthereof, or a pharmaceutically acceptable salt of an enantiomer thereof.19-20. (canceled) 21: The compound of claim 1, wherein said compoundbinds to APP and/or to the enzyme BACE and/or to an APP/BACE complex.22: The compound of claim 1, wherein said compound binds to APP andinhibits the enzyme BACE. 23: A pharmaceutical formulation comprising apharmaceutically acceptable carrier and a compound of claim
 1. 24-27.(canceled) 28: A method of preventing or delaying the onset of apre-Alzheimer's condition and/or cognitive dysfunction, and/orameliorating one or more symptoms of a pre-Alzheimer's condition and/orcognitive dysfunction, or preventing or delaying the progression of apre-Alzheimer's condition or cognitive dysfunction to Alzheimer'sdisease, said method comprising: administering to a subject in needthereof a compound of claim 1 in an amount sufficient to prevent ordelay the onset of a pre-Alzheimer's cognitive dysfunction, and/or toameliorate one or more symptoms of a pre-Alzheimer's cognitivedysfunction, and/or to prevent or delay the progression of apre-Alzheimer's cognitive dysfunction to Alzheimer's disease. 29: Themethod of claim 28, wherein said method is a method of: preventing ordelaying the transition from a cognitively asymptomatic pre-Alzheimer'scondition to a pre-Alzheimer's cognitive dysfunction; and/or preventingor delaying the onset of a pre-Alzheimer's cognitive dysfunction; and/orpreventing or delaying the progression of a pre-Alzheimer's cognitivedysfunction to Alzheimer's disease. 30-50. (canceled) 51: The method ofclaim 28, wherein: said administration produces a reduction in the CSFof levels of one or more components selected from the group consistingof Aβ42, sAPPβ, total-Tau (tTau), phospho-Tau (pTau), APPneo, solubleAβ40, pTau/Aβ42 ratio and tTau/Aβ42 ratio, and/or an increase in the CSFof levels of one or more components selected from the group consistingof Aβ42/Aβ40 ratio, Aβ42/Aβ38 ratio, sAPPα, sAPPα/sAPPβ ratio,sAPPα/Aβ40 ratio, and sAPPα/Aβ42 ratio; and/or said administrationproduces a reduction of the plaque load in the brain of the subject;and/or said administration produces a reduction in the rate of plaqueformation in the brain of the subject; and/or said administrationproduces an improvement in the cognitive abilities of the subject;and/or said administration produces an improvement in, a stabilizationof, or a reduction in the rate of decline of the clinical dementiarating (CDR) of the subject. 52-60. (canceled) 61: A method ofameliorating one or more symptoms of Alzheimer's disease, and/orreversing Alzheimer's disease, and/or reducing the rate of progressionof Alzheimer's disease, said method comprising: administering to asubject in need thereof a compound of claim 1 in an amount sufficient toameliorate one or more symptoms of Alzheimer's disease, and/or toreverse Alzheimer's disease, and/or to reduce the rate of progression ofAlzheimer's disease. 62-63. (canceled) 64: The method of claim 61,wherein: said subject is diagnosed with early stage Alzheimer's disease;or said subject is diagnosed with mid-stage Alzheimer's disease; or saidsubject is diagnosed with late-stage Alzheimer's disease. 65-67.(canceled) 68: The method of claim 61, wherein: said administeringameliorates one or more symptoms of Alzheimer's disease; and/or saidadministering reduces the rate of progression of Alzheimer's disease;and/or said administering results in a reduction in the CSF of levels ofone or more components selected from the group consisting of Aβ42,sAPPβ, total-Tau (tTau), phospho-Tau (pTau), APPneo, soluble Aβ40,pTau/Aβ42 ratio and tTau/Aβ42 ratio, and/or an increase in the CSF oflevels of one or more components selected from the group consisting ofAβ42/Aβ40 ratio, Aβ42/Aβ38 ratio, sAPPα, sAPPα/sAPPβ ratio, sAPPα/Aβ40ratio, and sAPPα/Aβ42 ratio; and/or said administration produces areduction of the plaque load in the brain of the subject; and/or saidadministration produces a reduction in the rate of plaque formation inthe brain of the subject; and/or said administration produces animprovement in the cognitive abilities of the subject; and/or saidadministration produces an improvement in, a stabilization of, or areduction in the rate of decline of the clinical dementia rating (CDR)of the subject; and/or said administration produces a perceivedimprovement in quality of life by the subject; and/or said administeringresults in reduced cerebral amyloidosis and/or downstreamneurodegeneration. 69-78. (canceled) 79: The method of claim 61,wherein: said subject shows a clinical dementia rating indicative ofAlzheimer's disease; and/or said subject has a familial risk for havingAlzheimer's disease; and/or said subject has a familial Alzheimer'sdisease (FAD) mutation. 80-93. (canceled) 94: A method of slowing theprogression, stopping, or reversing age-related macular degeneration(AMD) in a mammal, said method comprising administering to said mammal acompound of claim 1 in an amount sufficient to slow the progression,stop, or reverse age-related macular degeneration in said mammal. 95: Amethod for the treatment of a disease or disorder associated with BACEactivity in a subject in need thereof, wherein said method comprisesproviding to said subject a therapeutically effective amount of acompound of claim
 1. 96-97. (canceled) 98: A kit comprising one or morecontainers containing a compound of claim
 1. 99-101. (canceled)